Bacillus strains improving health and performance of production animals

ABSTRACT

The present invention relates to  Bacillus  strains which improves health and performance of production animals. The invention further relates to compositions comprising the  Bacillus  strains.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a 35 U.S.C. 371 national application ofinternational application no. PCT/US2016/014524 filed Jan. 22, 2016,which claims priority or the benefit under 35 U.S.C. 119 of U.S.application Nos. 62/106,892 and 62/260,882 filed Jan. 23, 2015 and Nov.30, 2015, respectively. The content of each application is fullyincorporated herein by reference.

REFERENCE TO A DEPOSIT OF BIOLOGICAL MATERIAL

This application contains a reference to a deposit of biologicalmaterial, which deposit is incorporated herein by reference.

REFERENCE TO SEQUENCE LISTING

This application contains a Sequence Listing in computer readable form.The computer readable form is incorporated herein by reference.

Index to sequence listing:

SEQ ID NO: 1 is 16S rDNA of DSM 29869.

SEQ ID NO: 2 is 16S rDNA of DSM 29870.

SEQ ID NO: 3 is 16S rDNA of DSM 29871.

SEQ ID NO: 4 is 16S rDNA of DSM 29872.

SEQ ID NO: 5 is 16S rDNA of Bacillus vallismortis from AB021198.

SEQ ID NO: 6 is 16S rDNA of Bacillus subtilis from AJ276351.

SEQ ID NO: 7 is 16S rDNA of Bacillus amyloliquefaciens from AB255669.

SEQ ID NO: 8 to SEQ ID NO: 13: PCR and sequencing primers.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention relates to Bacillus strains which improves thehealth and performance of production animals. The invention furtherrelates to compositions comprising the Bacillus strains.

Background of the Invention

Clostridium perfringens (C. perfringens) is a Gram-positive, rod-shaped,anaerobic, spore-forming bacterium of the genus Clostridium. C.perfringens is widely present in nature and can be found as a componentof decaying vegetation, marine sediment, the intestinal tract of humansand other vertebrates, insects, and soil.

Infections due to C. perfringens show evidence of tissue necrosis,bacteremia, emphysematous cholecystitis, and gas gangrene, which is alsoknown as clostridial myonecrosis. C. perfringens can also result inpolymicrobial anaerobic infections.

The incidence of Clostridium perfringens-associated necrotic enteritisin poultry has increased in countries that stopped using antibioticgrowth promoters. Necrotic enteritis is an enterotoxemic disease thatresults in significant economic losses in the poultry industry.

There is a need to develop tools and strategies for the prevention andcontrol of C. perfringens in mono-gastric animals such as poultry.Whilst the vaccination of animals has been suggested, there arechallenges associated with vaccinating thousands of animals. Thusdiscovering a solution which could be administered as an additive in ananimal feed would be advantageous.

It is thus an object of the invention to provide solutions whichprevents and/or controls C. perfringens in mono-gastric animals such aspoultry by use of an animal feed comprising one or more bacteria withactivity against Clostridium perfringens.

A further object of the invention is to provide a solution that does notpresent a risk for the health of the animal. The solution to thisproblem is use of non-hemolytic Bacillus strains with activity againstClostridium perfringens.

A challenge of delivering Bacillus spp. in feed is the common use ofantibiotics as growth promoters in feed. Therefore it is necessary todetermine the compatibility of strains with commonly-used feedantibiotics such as monensin in order to identify any potentialconflicts with use as a direct fed microbial. The present inventionrelates in one embodiment to a Bacillus strains with high compatibilitywith monensin.

Description of the Related Art

Knap et al. (2010) describes that Bacillus licheniformis has an effecton necrotic enteritis in broiler chickens (Knap, Lund, Kehlet, Hofacre,and Mathis; Bacillus licheniformis Prevents Necrotic Enteritis inBroiler Chickens; Avian Diseases 54(2):931-935. 2010). This Bacilluslicheniformis strain has no effect on Clostridium perfringens in vitroas demonstrated in Example 2.

Clostat (alias Bacillus PB6) is described in WO2007/064741. Bacillus PB6has antagonistic effect against C. perfringens. Bacillus PB6 ishemolytic as described in Example 1.

SUMMARY OF THE INVENTION

It has been surprisingly found that the addition of direct fed microbes(DFM) from Bacillus species to animal feed can be used to prevent and/orcontrol C. perfringens infections and/or necrotic enteritis inmono-gastric animal such as pigs and/or poultry. The Bacillus speciescan also improve the body weight gain and/or feed conversion rate (e.g.,in both Clostridium perfringens challenged and unchallenged chickens).

In a first aspect the invention relates to a Bacillus straincharacterized in that:

i) the Bacillus strain is non-hemolytic,

ii) the Bacillus strain has antimicrobial activity against Clostridiumperfringens and

iii) the Bacillus strain improves body weight gain and/or feedconversion rate in chickens fed with the Bacillus strain.

In a second aspect the invention relates to a Bacillus straincharacterized in that:

i) the Bacillus strain is non-hemolytic,

ii) the Bacillus strain has antimicrobial activity against Clostridiumperfringens and

iii) the Bacillus strain is sensitive to at least seven such as eight ofthe antibiotics selected from the group consisting of Vancomycin,Clindamycin, Chloramphenicol, Gentamicin, Kanamycin, Streptomycin,Erythromycin and Tetracycline.

In a third aspect the invention relates to a Bacillus straincharacterized in that:

i) the Bacillus strain is non-hemolytic,

ii) the Bacillus strain is sensitive to at least seven such as eight ofthe antibiotics selected from the group consisting of Vancomycin,Clindamycin, Chloramphenicol, Gentamicin, Kanamycin, Streptomycin,Erythromycin and Tetracycline and

iii) the Bacillus strain improves body weight gain and/or feedconversion rate in chickens fed with the Bacillus strain.

In a fourth aspect the invention relates to a Bacillus straincharacterized in that:

i) the Bacillus strain is non-hemolytic,

ii) the Bacillus strain has antimicrobial activity against Clostridiumperfringens and optionally

iii) the Bacillus strain has enzymatic activity under aerobic and/oranaerobic conditions that hydrolyzes one or more of the substratesselected from the group consisting of Amylose, Arabinan, Arabinoxylan,Casein and Xylan.

The invention further relates to the strain having the deposit accessionnumber DSM 29869; a strain having all of the identifying characteristicsof Bacillus DSM 29869 or a mutant thereof.

The invention further relates to the strain having the deposit accessionnumber DSM 29870; a strain having all of the identifying characteristicsof Bacillus DSM 29870 or a mutant thereof.

The invention further relates to the strain having the deposit accessionnumber DSM 29871; a strain having all of the identifying characteristicsof Bacillus DSM 29871 or a mutant thereof.

The invention further relates to the strain having the deposit accessionnumber DSM 29872; and a strain having all of the identifyingcharacteristics of Bacillus DSM 29872 or a mutant thereof.

The present invention also relates to compositions comprising one ormore of the Bacillus strains according to the invention which improveshealth and performance of production animals.

In a preferred embodiment the invention relates to a composition, e.g.,comprising a carrier and a Bacillus strain wherein:

i. the Bacillus strain is selected from the group consisting of:

-   -   the strain having the deposit accession number DSM 29869; a        strain having all of the identifying characteristics of Bacillus        DSM 29869 or a mutant thereof,    -   the strain having the deposit accession number DSM 29870; a        strain having all of the identifying characteristics of Bacillus        DSM 29870 or a mutant thereof,    -   the strain having the deposit accession number DSM 29871; a        strain having all of the identifying characteristics of Bacillus        DSM 29871 or a mutant thereof,    -   the strain having the deposit accession number DSM 29872; and a        strain having all of the identifying characteristics of Bacillus        DSM 29872 or a mutant thereof, and

ii. the Bacillus strain has antimicrobial activity against Clostridiumperfringens (such as Clostridium perfringens strains 23 or 48[Gholamiandekhordi A R, Ducatelle R, Heyndrickx M, Haesebrouck F, VanImmerseel F. 2006. Molecular and phenotypical characterization ofClostridium perfringens isolates from poultry flocks with differentdisease status. Vet. Microbiol. 113:143-152] and/or E. coli (such asATCC10536 or ATCC25922).

In a preferred embodiment the Bacillus strain has a high compatibilitywith monensin such as being compatible with at least 2.4 μg/ml monensinas determined in Example 12.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 Alignment of 16 S rDNA sequences. SEQ ID NO: 1 (DSM 29869), SEQID NO: 2 (DSM 29870), SEQ ID NO: 3 (DSM 29871), SEQ ID NO: 4 (DSM29872), SEQ ID NO: 5 (Bacillus vallismortis AB021198), SEQ ID NO: 6(Bacillus subtilis from AJ276351), and SEQ ID NO: 7 (Bacillusamyloliquefaciens AB255669) have been aligned. The region coveringposition 481-1200 is not shown in FIG. 1 (in this region all sequencesare identical).

DEFINITIONS

In general, the terms and phrases used herein have their art-recognizedmeaning, which can be found by reference to standard texts, journalreferences, and context known to those skilled in the art. The followingdefinitions are provided to clarify their specific use in context of thedisclosure.

As used herein, the singular forms “a”, “an” and “the” are intended toinclude the plural forms as well, unless the context clearly indicatesotherwise.

Antimicrobial activity against Clostridium perfringens: The term“Antimicrobial activity against Clostridium perfringens” means that thegrowth of Clostridium perfringens is inhibited and/or that some or allof the Clostridium perfringens are killed. This can be determined by theassay described in Example 2.

Body Weight Gain: The Body Weight Gain of an animal is the increase ofbody weight of the animal over a specified time period. An example ofBody Weight Gain determination is given in Example 8.

Composition: The term “composition” refers to a composition comprising acarrier and at least one bacterial strain as described herein. Thecompositions described herein may be mixed with an animal feed(s) andreferred to as a “mash feed.”

Control C. perfringens infections and/or necrotic enteritis: The term“control C. perfringens infections and/or necrotic enteritis” means amethod and/or composition that partly or completely inhibits C.perfringens infections and/or necrotic enteritis in an animal.Accordingly, the term “control C. perfringens infections and/or necroticenteritis” means the C. perfringens infections and/or the necroticenteritis is reduced or completely eliminated or prevented.

Direct Fed Microbial: The term “direct fed microbial” means livemicro-organisms including spores which, when administered in adequateamounts, confer a benefit, such as improved digestion or health, on thehost.

Enzymatic activity under aerobic conditions: The term “enzymaticactivity under aerobic conditions” means activity of enzymes produced bya Bacillus strain during growth under aerobic conditions as described inExample 6.

Enzymatic activity under anaerobic conditions: The term “enzymaticactivity under anaerobic conditions” means activity of enzymes producedby a Bacillus strain during growth under anaerobic conditions asdescribed in Example 6.

European Production Efficacy Factor (EPEF): The European ProductionEfficacy Factor is a way of comparing the live-bird performance offlocks. This single-figure facilitates comparison of performance withinand among farms and can be used to assess environmental, climatic andmanagemental variables. The EPEF is calculated as [(liveability(%)×Liveweight (kg))/(Age at depletion (days)×FCR)]×100, whereinlivability is the percentage of birds alive at slaughter, Liveweight isthe average weight of the birds at slaughter, age of depletion is theage of the birds at slaughter and FCR is the feed conversion ratio atslaughter.

Fed: The term “fed” means any type of oral administration such asadministration via an animal feed or via drinking water.

FCR (Feed Conversion Rate): FCR is a measure of an animal's efficiencyin converting feed mass into increases of the desired output. Animalsraised for meat—such as swine, poultry and fish—the output is the massgained by the animal. Specifically FCR is the mass of the food eatendivided by the output, all over a specified period. FCR can bedetermined as described in Example 8. Improvement in FCR means reductionof the FCR value. A FCR improvement of 2% means that the FCR was reducedby 2%.

Isolated: The term “isolated” means that the one or more bacterialstrains described herein are in a form or environment which does notoccur in nature, that is, the one or more bacterial strains are at leastpartially removed from one or more or all of the naturally occurringconstituents with which it is associated in nature.

Non-hemolytic: Hemolysis is the breakdown of red blood cells. Theability of bacterial colonies to induce hemolysis when grown on bloodagar is used to classify bacterial strains into hemolytic andnon-hemolytic strains. In this context hemolysis is defined as describedin EFSA Journal 2011; 9(11):2445, using Bacillus subtilis 168 (BGSC-1A1,Bacillus Genetic Stock Center) as a negative control. A Bacillus straincan be classified as non-hemolytic using the assay described in Example1.

Poultry: The term “poultry” means domesticated birds kept by humans forthe eggs they produce and/or their meat and/or their feathers. Poultryincludes broilers and layers. Poultry include members of the superorderGalloanserae (fowl), especially the order Galliformes (which includeschickens, Guineafowls, quails and turkeys) and the family Anatidae, inorder Anseriformes, commonly known as “waterfowl” and including domesticducks and domestic geese. Poultry also includes other birds that arekilled for their meat, such as the young of pigeons. Examples of poultryinclude chickens (including layers, broilers and chicks), ducks, geese,pigeons, turkeys and quail.

Prevent C. perfringens infections and/or necrotic enteritis: The term“prevent C. perfringens infections and/or necrotic enteritis” means amethod and/or composition that prevents development of a C. perfringensinfection and/or necrotic enteritis in an animal.

Sensitive to antibiotics: The term “sensitive to antibiotics” means thephenotypic property of a bacterial strain, that growth of said bacterialstrain is inhibited under conditions where the bacterial strain wouldotherwise grow. In this context sensitivity to antibiotics is testedafter the CLSI guidelines (M07-A9 Methods for Dilution AntimicrobialSusceptibility Tests for Bacteria That Grow Aerobically; 2012). The S.aureus ATCC 29213 is used as reference strain, which means that itshould be included in the test, and that the results are only valid ifS. aureus ATCC 29213 show results in compliance with the breakpoints ofthe CLSI guideline (see Example 5 Table 5.5) (M100-S24 PerformanceStandards for Antimicrobial Susceptibility Testing; informationalSupplement, 2014). A strain of Bacillus is considered sensitive if thegrowth is detected at or below the breakpoint concentration of theappropriate antibiotic specified in EFSA journal 2012; 10(6):2740.

Spore: The terms “spore” and “endospore” are interchangeable and havetheir normal meaning which is well known and understood by those ofskill in the art. As used herein, the term spore refers to amicroorganism in its dormant, protected state.

Stable: The term “stable” is a term that is known in the art, and in apreferred aspect, stable is intended to mean the ability of themicroorganism to remain in a spore form until it is administered to ananimal to improve the health of the animal.

Swine: The term “swine” or “pigs” means domesticated pigs kept by humansfor food, such as their meat. Swine includes members of the genus Sus,such as Sus scrofa domesticus or Sus domesticus and include piglets,growing pigs, and sows.

DETAILED DESCRIPTION OF THE INVENTION

It has been surprisingly found that the addition of direct fed microbes(DFM) from Bacillus species to animal feed can be used to prevent and/orcontrol C. perfringens infections and/or necrotic enteritis inmono-gastric animal such as pigs and/or poultry and at the same timeimprove the body weight gain and/or feed conversion rate (in bothClostridium perfringens challenged and unchallenged chickens).

Bacillus Strains of the Invention

The invention relates to the following aspects with respect to Bacillusstrains:

Aspect 1: A Bacillus strain characterized in that:

i) the Bacillus strain is non-hemolytic,

ii) the Bacillus strain has antimicrobial activity against Clostridiumperfringens and

iii) the Bacillus strain improves body weight gain and/or feedconversion rate in chickens fed with the Bacillus strain.

Aspect 2: A Bacillus strain characterized in that:

i) the Bacillus strain is non-hemolytic,

ii) the Bacillus strain has antimicrobial activity against Clostridiumperfringens and

iii) the Bacillus strain is sensitive to at least seven such as eight ofthe antibiotics selected from the group consisting of Vancomycin,Clindamycin, Chloramphenicol, Gentamicin, Kanamycin, Streptomycin,Erythromycin and Tetracycline.

Aspect 3: A Bacillus strain characterized in that:

i) the Bacillus strain is non-hemolytic,

ii) the Bacillus strain is sensitive to at least seven such as eight ofthe antibiotics selected from the group consisting of Vancomycin,Clindamycin, Chloramphenicol, Gentamicin, Kanamycin, Streptomycin,Erythromycin and Tetracycline and

iii) the Bacillus strain improves body weight gain and/or feedconversion rate in chickens fed with the Bacillus strain.

Aspect 4: A Bacillus strain characterized in that:

i) the Bacillus strain is non-hemolytic,

ii) the Bacillus strain has antimicrobial activity against Clostridiumperfringens and optionally

iii) the Bacillus strain has enzymatic activity under aerobic and/oranaerobic conditions that hydrolyzes one or more of the substratesselected from the group consisting of Amylose, Arabinan, Arabinoxylan,Casein and Xylan.

In a preferred embodiment the Bacillus according to Aspect 1, 2 3 or 4has a high compatibility with monensin such as being compatible with atleast 2.3 μg/ml monensin as determined in Example 12. It is even morepreferred that the Bacillus strain is compatible with at least 2.4 μg/mlmonensin as determined in Example 12 (such as at least 2.5 μg/mlmonensin as determined in Example 12, such as at least 2.6 μg/mlmonensin as determined in Example 12 or such as at least 2.7 μg/mlmonensin as determined in Example 12).

Sensitivity to Vancomycin, Clindamycin, Chloramphenicol, Gentamicin,Kanamycin, Streptomycin, Erythromycin and Tetracycline can, e.g., bedetermined as described in Example 5.

Enzymatic activity under aerobic and/or anaerobic conditions thathydrolyzes one or more of the substrates selected from the groupconsisting of Amylose, Arabinan, Arabinoxylan, Casein and Xylan can,e.g., be determined as described in Example 6.

In one embodiment the Bacillus strain according to Aspect 1, 2 or 3 hasenzymatic activity under aerobic and/or anaerobic conditions thathydrolyzes one or more of the substrates selected from the groupconsisting of Amylose, Arabinan, Arabinoxylan, Casein and Xylan.

In one embodiment the Bacillus strain according to Aspect 2, 3 or 4improves one or more performance parameters in poultry selected from thelist consisting of body weight gain, European Production Efficacy Factorand feed conversion rate in chickens fed with the Bacillus strain.

In one embodiment, the Bacillus strain according to Aspect 3 hasantimicrobial activity against Clostridium perfringens. Antimicrobialactivity against Clostridium perfringens can, e.g., be determined asdescribed in Example 2.

In one embodiment the Bacillus strain according to Aspect 1 or 4 issensitive to at least seven such as eight of the antibiotics selectedfrom the group consisting of Vancomycin, Clindamycin, Chloramphenicol,Gentamicin, Kanamycin, Streptomycin, Erythromycin and Tetracycline.

In one embodiment the improvement of feed conversion rate (FCR) forAspect 1, 2, 3 or 4 results in a FCR of −2.5% or less than −2.5%, suchas less than −2.6%, such as less than −2.7%, such as less than −2.8%,such less than −2.9%, such as less than −3.0%. In a preferred embodimentthe improvement of FCR results in a FCR of from −5% to −2% such as a FCRfrom −4% to −2%, such as a FCR of from −3.5% to −2.5%. In a specificembodiment the improvement of FCR for Aspect 1, 2, 3 or 4 results in aFCR within an interval selected from the group consisting of from −5% to−4.5%, from −4.5% to −4%, from −4% to −3.8%, from −3.8% to −3.6%, from−3.6% to −3.4%, from −3.4% to −3.2%, from −3.2 to −3.0%, from −3.0% to−2.8% and from −2.8 to −2.5%, or any combination of these intervals. TheFCR can be determined as described in Example 8.

In one embodiment the improvement in body weight gain for Aspect 1, 2, 3or 4 results in a body weight gain of at least 0.5%, such as at least0.8%, such as at least 1.5%, such as at least 1.8%, such as at least2.0%, such as at least 2.3%, such as at least 3.5%, such as at least4.2%, such as at least 5.2%, such as at least 6.5%, such as at least7.3%. In a preferred embodiment the improvement in body weight gain forAspect 1, 2, 3 or 4 results in a body weight gain selected from thegroup consisting of from 1.8% to 2.0%, from 2.0% to 2.2%, from 2.2% to2.4%, from 2.4% to 2.6%, from 2.6% to 2.8%, from 2.8% to 3.0%, from 3.0%to 3.2%, from 3.2% to 3.4%, from 3.4% to 3.6%, from 3.6% to 3.8%, from3.8% to 4.0%, from 4% to 5%, from 5% to 7%, from 7% to 10%, or anycombination thereof. The body weight gain can be determined as describedin Example 8.

In one embodiment the Bacillus strain according to Aspect 1, 2, 3 or 4wherein the Bacillus strain comprises 16S rDNA that is more than 98%(such as more than 98.5%, such as more than 99%, such as more than99.5%, such as more than 99.5%) sequence identity to SEQ ID NO: 1.

In one embodiment the Bacillus strain according to Aspect 1, 2, 3 or 4wherein the Bacillus strain comprises 16S rDNA that is more than 98%(such as more than 98.5%, such as more than 99%, such as more than99.5%) sequence identity to SEQ ID NO: 2.

In one embodiment the Bacillus strain according to Aspect 1, 2, 3 or 4wherein the Bacillus strain comprises 16S rDNA that is more than 98%(such as more than 98.5%, such as more than 99%, such as more than99.5%) sequence identity to SEQ ID NO: 3.

In one embodiment the Bacillus strain according to Aspect 1, 2, 3 or 4wherein the Bacillus strain comprises 16S rDNA that is more than 98%(such as more than 98.5%, such as more than 99%, such as more than99.5%) sequence identity to SEQ ID NO: 4.

In one embodiment the Bacillus strain according to Aspect 1, 2, 3 or 4is a Bacillus subtilis strain, a Bacillus amyloliquefaciens strain or aBacillus licheniformis strain.

In one embodiment the Bacillus strain according to Aspect 1, 2, 3 or 4has antimicrobial effect against E. coli. The effect against E. colican, e.g., be determined as described in Example 4.

In one embodiment the Bacillus strain according to Aspect 1, 2, 3 or 4is the Bacillus strain having deposit accession number DSM 29869, or astrain having all of the identifying characteristics of Bacillus DSM29869 or a mutant thereof that has antimicrobial activity againstClostridium perfringens. In another embodiment the Bacillus strainaccording to Aspect 1, 2, 3 or 4 is the Bacillus strain having depositaccession number DSM 29870, or a strain having all of the identifyingcharacteristics of Bacillus DSM 29870 or a mutant thereof that hasantimicrobial activity against Clostridium perfringens. In yet anotherembodiment the Bacillus strain according to Aspect 1, 2, 3 or 4 is theBacillus strain having deposit accession number DSM 29871, or a strainhaving all of the identifying characteristics of Bacillus DSM 29871 or amutant thereof that has antimicrobial activity against Clostridiumperfringens. In a further embodiment the Bacillus strain according toAspect 1, 2, 3 or 4 is the Bacillus strain having deposit accessionnumber DSM 29872, or a strain having all of the identifyingcharacteristics of Bacillus DSM 29872 or a mutant thereof that hasantimicrobial activity against Clostridium perfringens.

The invention relates in one embodiment to a Bacillus strain havingdeposit accession number DSM 29869 or a strain having all of theidentifying characteristics of Bacillus DSM 29869 or a mutant thereof.The invention relates in another embodiment to a Bacillus strain havingdeposit accession number DSM 29870 or a strain having all of theidentifying characteristics of Bacillus DSM 29870 or a mutant thereof.The invention relates in another embodiment to a Bacillus strain havingdeposit accession number DSM 29871 or a strain having all of theidentifying characteristics of Bacillus DSM 29871 or a mutant thereof.The invention relates in a further embodiment to a Bacillus strainhaving deposit accession number DSM 29872 or a strain having all of theidentifying characteristics of Bacillus DSM 29872 or a mutant thereof.

The invention also relates to a biologically pure culture of theBacillus strain according to aspect 1, 2, 3 or 4. In a furtherembodiment the invention relates to a biologically pure culture of theBacillus strain having deposit accession number DSM 29869 or a strainhaving all of the identifying characteristics of Bacillus DSM 29869 or amutant thereof. In a further embodiment the invention relates to abiologically pure culture of the Bacillus strain having depositaccession number DSM 29870 or a strain having all of the identifyingcharacteristics of Bacillus DSM 29870 or a mutant thereof. In a furtherembodiment the invention relates to a biologically pure culture of theBacillus strain having deposit accession number DSM 29871 or a strainhaving all of the identifying characteristics of Bacillus DSM 29871 or amutant thereof. In a further embodiment the invention relates to abiologically pure culture of the Bacillus strain having depositaccession number DSM 29872 or a strain having all of the identifyingcharacteristics of Bacillus DSM 29872 or a mutant thereof.

The invention also relates to an isolated Bacillus strain according toaspect 1, 2, 3 or 4. In a further embodiment the invention also relatesto an isolated Bacillus strain having deposit accession number DSM 29869or an isolated strain having all of the identifying characteristics ofBacillus DSM 29869 or a mutant thereof. In a further embodiment theinvention also relates to an isolated Bacillus strain having depositaccession number DSM 29870 or an isolated strain having all of theidentifying characteristics of Bacillus DSM 29870 or a mutant thereof.In a further embodiment the invention also relates to an isolatedBacillus strain having deposit accession number DSM 29871 or an isolatedstrain having all of the identifying characteristics of Bacillus DSM29871 or a mutant thereof. In a further embodiment the invention alsorelates to an isolated Bacillus strain having deposit accession numberDSM 29872 or an isolated strain having all of the identifyingcharacteristics of Bacillus DSM 29872 or a mutant thereof.

Compositions of the Invention

The invention relates to a composition comprising spores of one or moreBacillus strains according to invention.

More specifically the invention relates to the following aspects withrespect to compositions comprising Bacillus strains:

Aspect 5: A composition comprising spores of a Bacillus straincharacterized in that:

i) the Bacillus strain is non-hemolytic,

ii) the Bacillus strain has antimicrobial activity against Clostridiumperfringens and

iii) the Bacillus strain improves body weight gain and/or feedconversion rate in chickens fed with the Bacillus strain.

Aspect 6: A composition comprising spores of a Bacillus straincharacterized in that:

i) the Bacillus strain is non-hemolytic,

ii) the Bacillus strain has antimicrobial activity against Clostridiumperfringens and

iii) the Bacillus strain is sensitive to at least seven such as eight ofthe antibiotics selected from the group consisting of Vancomycin,Clindamycin, Chloramphenicol, Gentamicin, Kanamycin, Streptomycin,Erythromycin and Tetracycline.

Aspect 7: A composition comprising spores of a Bacillus straincharacterized in that:

i) the Bacillus strain is non-hemolytic,

ii) the Bacillus strain is sensitive to at least seven such as eight ofthe antibiotics selected from the group consisting of Vancomycin,Clindamycin, Chloramphenicol, Gentamicin, Kanamycin, Streptomycin,Erythromycin and Tetracycline and

iii) the Bacillus strain improves body weight gain and/or feedconversion rate in chickens fed with the Bacillus strain.

Aspect 8: A composition comprising spores of a Bacillus straincharacterized in that:

i) the Bacillus strain is non-hemolytic,

ii) the Bacillus strain has antimicrobial activity against Clostridiumperfringens and optionally

iii) the Bacillus strain has enzymatic activity under aerobic and/oranaerobic conditions that hydrolyzes one or more of the substratesselected from the group consisting of Amylose, Arabinan, Arabinoxylan,Casein and Xylan.

In a preferred embodiment the Bacillus strain(s) of the compositionaccording to Aspect 5, 6, 7 or 8 has a high compatibility with monensinsuch as being compatible with at least 2.3 μg/ml monensin as determinedin Example 12. It is even more preferred that the Bacillus strain iscompatible with at least 2.4 μg/ml monensin as determined in Example 12(such as at least 2.5 μg/ml monensin as determined in Example 12, suchas at least 2.6 μg/ml monensin as determined in Example 12 or such as atleast 2.7 μg/ml monensin as determined in Example 12).

Sensitivity to Vancomycin, Clindamycin, Chloramphenicol, Gentamicin,Kanamycin, Streptomycin, Erythromycin and Tetracycline can, e.g., bedetermined as described in Example 5.

Enzymatic activity under aerobic and/or anaerobic conditions thathydrolyzes one or more of the substrates selected from the groupconsisting of Amylose, Arabinan, Arabinoxylan, Casein and Xylan can,e.g., be determined as described in Example 6.

In one embodiment the Bacillus strain of the composition according toAspect 5, 6 or 7 has enzymatic activity under aerobic and/or anaerobicconditions that hydrolyzes one or more of the substrates selected fromthe group consisting of Amylose, Arabinan, Arabinoxylan, Casein andXylan.

In one embodiment the Bacillus strain of the composition according toAspect 6, 7, or 8 improves one or more performance parameters in poultryselected from the list consisting of body weight gain, EuropeanProduction Efficacy Factor and feed conversion rate in chickens fed withthe Bacillus strain.

In one embodiment the Bacillus strain of the composition according toAspect 8 has antimicrobial activity against Clostridium perfringens.Antimicrobial activity against Clostridium perfringens can, e.g., bedetermined as described in Example 2.

In one embodiment the Bacillus strain of the composition according toAspect 5 or 8 is sensitive to at least seven such as eight of theantibiotics selected from the group consisting of Vancomycin,Clindamycin, Chloramphenicol, Gentamicin, Kanamycin, Streptomycin,Erythromycin and Tetracycline.

In one embodiment the improvement of feed conversion rate (FCR) forAspect 5, 6, 7 or 8 results in a FCR of −2.5% or less than −2.5%, suchas less than −2.6%, such as less than −2.7%, such as less than −2.8%,such less than −2.9%, such as less than −3.0%. In a preferred embodimentthe improvement of FCR results in a FCR of from −5% to −2% such as a FCRfrom −4% to −2%, such as a FCR of from −3.5% to −2.5%. In a specificembodiment the improvement of FCR for Aspect 1, 2, 3 or 4 results in aFCR within an interval selected from the group consisting of from −5% to−4.5%, from −4.5% to −4%, from −4% to −3.8%, from −3.8% to −3.6%, from−3.6% to −3.4%, from −3.4% to −3.2%, from −3.2 to −3.0%, from −3.0% to−2.8% and from −2.8 to −2.5%, or any combination of these intervals. TheFCR can be determined as described in Example 8.

In one embodiment the improvement in body weight gain for Aspect 5, 6, 7or 8 results in a body weight gain of at least 0.5%, such as at least0.8%, such as at least 1.5%, such as at least 1.8%, such as at least2.0%, such as at least 2.3%, such as at least 3.5%, such as at least4.2%, such as at least 5.2%, such as at least 6.5%, such as at least7.3%. In a preferred embodiment the improvement in body weight gain forAspect 1, 2, 3 or 4 results in a body weight gain selected from thegroup consisting of from 1.8% to 2.0%, from 2.0% to 2.2%, from 2.2% to2.4%, from 2.4% to 2.6%, from 2.6% to 2.8%, from 2.8% to 3.0%, from 3.0%to 3.2%, from 3.2% to 3.4%, from 3.4% to 3.6%, from 3.6% to 3.8%, from3.8% to 4.0%, from 4% to 5%, from 5% to 7%, from 7% to 10%, or anycombination thereof. The body weight gain can be determined as describedin Example 8.

In one embodiment the Bacillus strain of the composition according toAspect 5, 6, 7 or 8 comprises 16S rDNA that has more than 98% (such asmore than 98.5%, such as more than 99%, such as more than 99.5%)sequence identity to SEQ ID NO: 1 and/or more than 98% (such as morethan 98.5%, such as more than 99%, such as more than 99.5%) sequenceidentity to SEQ ID NO: 2 and/or more than 98% (such as more than 98.5%,such as more than 99%, such as more than 99.5%) sequence identity to SEQID NO: 3 and/or more than 98% (such as more than 98.5%, such as morethan 99%, such as more than 99.5%) sequence identity to SEQ ID NO: 4.

In one embodiment the Bacillus strain of the composition according toAspect 5, 6, 7 or 8 is a Bacillus subtilis strain, or a Bacillusamyloliquefaciens strain.

In one embodiment the Bacillus strain of the composition according toAspect 5, 6, 7 or 8 has antimicrobial effect against E. coli. The effectagainst E. coli can, e.g., be determined as described in Example 4.

In one embodiment the Bacillus strain of the composition according toAspect 5, 6, 7 or 8 is the Bacillus strain having deposit accessionnumber DSM 29869, or a strain having all of the identifyingcharacteristics of Bacillus DSM 29869 or a mutant thereof that hasantimicrobial activity against Clostridium perfringens. In anotherembodiment the Bacillus strain of the composition according to Aspect 5,6, 7 or 8 is the Bacillus strain having deposit accession number DSM29870, or a strain having all of the identifying characteristics ofBacillus DSM 29870 or a mutant thereof that has antimicrobial activityagainst Clostridium perfringens. In yet another embodiment the Bacillusstrain of the composition according to Aspect 5, 6, 7 or 8 is theBacillus strain having deposit accession number DSM 29871, or a strainhaving all of the identifying characteristics of Bacillus DSM 29871 or amutant thereof that has antimicrobial activity against Clostridiumperfringens. In a further embodiment the Bacillus strain of thecomposition according to Aspect 5, 6, 7 or 8 is the Bacillus strainhaving deposit accession number DSM 29872, or a strain having all of theidentifying characteristics of Bacillus DSM 29872 or a mutant thereofthat has antimicrobial activity against Clostridium perfringens.

The invention relates in one embodiment to a composition comprising aBacillus having deposit accession number DSM 29869 or a strain havingall of the identifying characteristics of Bacillus DSM 29869 or a mutantthereof. The invention relates in another embodiment to a compositioncomprising a Bacillus having deposit accession number DSM 29870 or astrain having all of the identifying characteristics of Bacillus DSM29870 or a mutant thereof. The invention relates in another embodimentto a composition comprising a Bacillus having deposit accession numberDSM 29871 or a strain having all of the identifying characteristics ofBacillus DSM 29871 or a mutant thereof. The invention relates in afurther embodiment to a composition comprising a Bacillus having depositaccession number DSM 29872 or a strain having all of the identifyingcharacteristics of Bacillus DSM 29872 or a mutant thereof.

In one embodiment of Aspect 5, 6, 7, or 8 the bacillus spores of thecomposition are present as dried spores such as spray-dried spores. Inone embodiment of Aspect 5, 6, 7, or 8 the bacillus spores of thecomposition are present as stable spores. The composition according toAspect 5, 6, 7, or 8 can also be a liquid composition and/or compriseculture supernatant comprising one or more Bacillus strain(s) of theinvention.

In one embodiment of Aspect 5, 6, 7, or 8 the composition furthercomprises a carrier. The carrier can comprise one or more of thefollowing compounds: water, glycerol, ethylene glycol, 1, 2-propyleneglycol or 1, 3-propylene glycol, sodium chloride, sodium benzoate,potassium sorbate, sodium sulfate, potassium sulfate, magnesium sulfate,sodium thiosulfate, calcium carbonate, sodium citrate, dextrin,maltodextrin, glucose, sucrose, sorbitol, lactose, wheat flour, wheatbran, corn gluten meal, starch, cellulose farigel, cassava cores, sodiumaluminium silicate, colloidal amorphous silica, Sipernat 50S,polyethylene glycol 200, polyethylene glycol 400, polyethylene glycol600, polyethylene glycol 1000, polyethylene glycol 1500, polyethyleneglycol 4000 and carbopol.

In a preferred embodiment of Aspect 5, 6, 7, or 8 the compositionfurther comprises calcium carbonate and sodium aluminium silicate.

In a preferred embodiment of Aspect 5, 6, 7, or 8 the compositionfurther comprises calcium carbonate, sodium aluminium silicate andsucrose.

In another preferred embodiment of Aspect 5, 6, 7, or 8 the compositionfurther comprises one or more carriers such as one or more carriersselected from the group consisting of Calcium carbonate, sodium sulfate,starch, farigel and cassava cores.

In another preferred embodiment of Aspect 5, 6, 7, or 8 the compositionfurther comprises one or more flowability agents such as sodiumaluminium silicate and/or colloidal amorphous silica (e.g., Sipernat50S).

In another preferred embodiment of Aspect 5, 6, 7, or 8 the compositionfurther comprises one or more binder such as one or more bindersselected from the group consisting of sucrose, sorbitol, glycerol,polyethylene glycol 200, polyethylene glycol 400, polyethylene glycol600, polyethylene glycol 1000, polyethylene glycol 1500, polyethyleneglycol 4000, dextrin, maltodextrin and carbopol.

In a preferred embodiment the composition comprises Bacillus DSM 29869,calcium carbonate and sodium aluminium silicate.

In a preferred embodiment the composition comprises Bacillus DSM 29869,calcium carbonate, sodium aluminium silicate and sucrose.

In a preferred embodiment the composition comprises Bacillus DSM 29869and one or more carriers such as one or more carriers selected from thegroup consisting of calcium carbonate, sodium sulphate, starch, farigeland cassava cores.

In a preferred embodiment the composition comprises Bacillus DSM 29869and one or more flowability agents such as sodium aluminium silicateand/or colloidal amorphous silica (e.g., Sipernat 50S).

In a preferred embodiment the composition comprises Bacillus DSM 29869and one or more binder such as one or more binders selected from thegroup consisting of sucrose, sorbitol, glycerol, polyethylene glycol200, polyethylene glycol 400, polyethylene glycol 600, polyethyleneglycol 1000, polyethylene glycol 1500, polyethylene glycol 4000,dextrin, maltodextrin and carbopol.

In a preferred embodiment the composition comprises Bacillus DSM 29870,calcium carbonate and sodium aluminium silicate.

In a preferred embodiment the composition comprises Bacillus DSM 29870,calcium carbonate, sodium aluminium silicate and sucrose.

In a preferred embodiment the composition comprises Bacillus DSM 29870and one or more carriers such as one or more carriers selected from thegroup consisting of calcium carbonate, sodium sulphate, starch, farigeland cassava cores.

In a preferred embodiment the composition comprises Bacillus DSM 29870and one or more flowability agents such as sodium aluminium silicateand/or colloidal amorphous silica (e.g., Sipernat 50S).

In a preferred embodiment the composition comprises Bacillus DSM 29870and one or more binder such as one or more binders selected from thegroup consisting of sucrose, sorbitol, glycerol, polyethylene glycol200, polyethylene glycol 400, polyethylene glycol 600, polyethyleneglycol 1000, polyethylene glycol 1500, polyethylene glycol 4000,dextrin, maltodextrin and carbopol.

In a preferred embodiment the composition comprises Bacillus DSM 29871,calcium carbonate and sodium aluminium silicate.

In a preferred embodiment the composition comprises Bacillus DSM 29871,calcium carbonate, sodium aluminium silicate and sucrose.

In a preferred embodiment the composition comprises Bacillus DSM 29871and one or more carriers such as one or more carriers selected from thegroup consisting of calcium carbonate, sodium sulphate, starch, farigeland cassava cores.

In a preferred embodiment the composition comprises Bacillus DSM 29871and one or more flowability agents such as sodium aluminium silicateand/or colloidal amorphous silica (e.g., Sipernat 50S).

In a preferred embodiment the composition comprises Bacillus DSM 29871and one or more binder such as one or more binders selected from thegroup consisting of sucrose, sorbitol, glycerol, polyethylene glycol200, polyethylene glycol 400, polyethylene glycol 600, polyethyleneglycol 1000, polyethylene glycol 1500, polyethylene glycol 4000,dextrin, maltodextrin and carbopol.

In a preferred embodiment the composition comprises Bacillus DSM 29872,calcium carbonate and sodium aluminium silicate.

In a preferred embodiment the composition comprises Bacillus DSM 29872,calcium carbonate, sodium aluminium silicate and sucrose.

In a preferred embodiment the composition comprises Bacillus DSM 29872and one or more carriers such as one or more carriers selected from thegroup consisting of calcium carbonate, sodium sulphate, starch, farigeland cassava cores.

In a preferred embodiment the composition comprises Bacillus DSM 29872and one or more flowability agents such as sodium aluminium silicateand/or colloidal amorphous silica (e.g., Sipernat 50S).

In a preferred embodiment the composition comprises Bacillus DSM 29872and one or more binder such as one or more binders selected from thegroup consisting of sucrose, sorbitol, glycerol, polyethylene glycol200, polyethylene glycol 400, polyethylene glycol 600, polyethyleneglycol 1000, polyethylene glycol 1500, polyethylene glycol 4000,dextrin, maltodextrin and carbopol.

In one embodiment the composition comprises one or more coccidiostatswherein the composition is, e.g., a premix.

In a preferred embodiment the composition according to Aspect 5, 6, 7,or 8 the composition comprises from 10⁵ to 10¹⁸ CFU/g of isolatedBacillus spores.

In a further embodiment, the composition according to the inventioncomprises one or more bacterial strains such as at least two of theabove strains up to and including all of the strains in the groupconsisting of DSM 29869, DSM 29870, DSM 29871 and DSM 29872.

In an embodiment to any of the aforementioned embodiments the Bacillusspore kills/inhibits at least 40% (such as at least 45%, at least 50%,at least 60%, at least 70% or at least 80%) of Clostridium perfringensafter, e.g., 24 hours, e.g., determined as described in Example 2.

In another embodiment of the invention the composition further comprisesone or more additional microbes. In another embodiment of the inventionthe composition further comprises one or more enzymes. In anotherembodiment of the invention the composition further comprises one ormore vitamins. In another embodiment of the invention the compositionfurther comprises one or more minerals. In another embodiment of theinvention the composition further comprises one or more amino acids. Inanother embodiment of the invention the composition further comprisesone or more other feed ingredients.

In an embodiment to any of the aforementioned embodiments, thecomposition also improves the health of the mono-gastric animal when fedto said animal. In another embodiment to any of the aforementionedembodiments, the composition also increases the egg yield of poultrywhen fed said poultry. In an embodiment to any of the aforementionedembodiments, the composition increases the meat yield of themono-gastric animal when fed to said animal.

In a preferred embodiment, the composition comprises one or morebacterial strains described herein, wherein the bacterial count of eachof the bacterial strains is between 1×10⁴ and 1×10¹⁸ CFU/kg ofcomposition, preferably between 1×10⁷ and 1×10¹⁶ CFU/kg of composition,more preferably between 1×10¹⁰ and 1×10¹⁵ CFU/kg of composition and mostpreferably between 1×10¹¹ and 1×10¹⁴ CFU/kg of composition.

In a preferred embodiment, the bacterial count of each of the bacterialstrains in the composition is between 1×10⁴ and 1×10¹⁸ CFU/kg of drymatter, preferably between 1×10⁷ and 1×10¹⁶ CFU/kg of dry matter, morepreferably between 1×10¹⁰ and 1)(10¹⁵ CFU/kg of dry matter and mostpreferably between 1×10¹¹ and 1×10¹⁴ CFU/kg of dry matter. In a morepreferred embodiment the bacterial count of each of the bacterialstrains in the animal feed is between 1×10⁸ and 1×10¹⁰ CFU/kg of drymatter.

In a preferred embodiment, the composition has a bacterial count of eachBacillus spore between 1×10³ and 1×10¹³ CFU/animal/day, preferablybetween 1×10⁵ and 1×10¹¹ CFU/animal/day, more preferably between 1×10⁶and 1×10¹⁰ CFU/animal/day and most preferably between 1×10⁷ and 1×10⁹CFU/animal/day.

In still yet another embodiment of the invention, the one or morebacterial strains are present in the composition in form of a spore suchas a stable spore. In still a further embodiment of the invention, thestable spore will germinate in the intestine and/or stomach of themono-gastric animal.

In one embodiment, the one or more bacterial strains are stable whensubjected to pressures applied/achieved during an extrusion process forpelleting. In a particular embodiment, the one or more bacterial strainsare stable at pressures ranging from 1 bar to 40 bar, particularly 10bar to 40 bar, more particularly 15 bar to 40 bar, even moreparticularly 20 bar to 40 bar, still even more particularly 35 bar to 37bar, even still more particularly 36 bar.

In a particular embodiment, the one or more bacterial strains are stableat high temperatures. In particular, the bacterial strains are stablewhen they are subjected to temperatures achieved during an extrusionprocess for pelleting. In an even more particular embodiment, the one ormore bacterial strains are stable at temperatures ranging from 80° C. to120° C., particularly temperatures ranging from, 90° C. to 120° C., evenmore particularly temperatures ranging from 95° C. to 120° C.

In another aspect, the invention relates to a composition comprising acarrier and one or more of the bacteria cultures having characteristicssubstantially identical to that of a strain selected from the groupconsisting of:

the strain having the deposit accession number DSM 29869;

the strain having the deposit accession number DSM 29870;

the strain having the deposit accession number DSM 29871; and

the strain having the deposit accession number DSM 29872; or

any combination thereof.

In an embodiment, the composition comprises a carrier and the strainhaving the deposit accession number DSM 29869, or a strain having all ofthe identifying characteristics of Bacillus DSM 29869 or a mutantthereof.

In an embodiment, the composition comprises a carrier and the strainhaving the deposit accession number DSM 29870, or a strain having all ofthe identifying characteristics of Bacillus DSM 29870 or a mutantthereof.

In an embodiment, the composition comprises a carrier and the strainhaving the deposit accession number DSM 29871, or a strain having all ofthe identifying characteristics of Bacillus DSM 29871 or a mutantthereof.

In an embodiment, the composition comprises a carrier and the strainhaving the deposit accession number DSM 29872, or a strain having all ofthe identifying characteristics of Bacillus DSM 29872 or a mutantthereof.

In an embodiment, the composition further comprises one or moreadditional microbes. In a particular embodiment, the composition furthercomprises a bacterium from one or more of the following genera:Lactobacillus, Lactococcus, Streptococcus, Bacillus, Pediococcus,Enterococcus, Leuconostoc, Carnobacterium, Propionibacterium,Bifidobacterium, Clostridium and Megasphaera or any combination thereof.

In a particular embodiment, the composition further comprises abacterium from one or more of the following strains of Bacillusamyloliquefaciens, Bacillus subtilis, Bacillus pumilus, Bacilluspolymyxa, Bacillus licheniformis, Bacillus megaterium, Bacilluscoagulans, Bacillus circulans, or any combination thereof.

In a particular embodiment, the composition further comprises one ormore types of yeast. The one or more types of yeast can be selected fromthe group consisting of Saccharomycetaceae, Saccharomyces (such as S.cerevisiae and/or S. boulardii), Kluyveromyces (such as K. marxianus andK. lactis), Candida (such as C. utilis, also called Torula yeast),Pichia (such as P. pastoris), Torulaspora (such as T. delbrueckii),Phaffia yeasts and Basidiomycota.

In an embodiment to any of the aforementioned embodiments thecomposition further comprises a carrier. The carrier can comprise one ormore of the following compounds: water, glycerol, ethylene glycol, 1,2-propylene glycol or 1, 3-propylene glycol, sodium chloride, sodiumbenzoate, potassium sorbate, sodium sulfate, potassium sulfate,magnesium sulfate, sodium thiosulfate, calcium carbonate, sodiumcitrate, dextrin, maltodextrin, glucose, sucrose, sorbitol, lactose,wheat flour, wheat bran, corn gluten meal, starch, cellulose, farigel,cassava cores, sodium aluminium silicate, colloidal amorphous silica,Sipernat 50S, polyethylene glycol 200, polyethylene glycol 400,polyethylene glycol 600, polyethylene glycol 1000, polyethylene glycol1500, polyethylene glycol 4000 and carbopol.

In another embodiment, the composition described herein can optionallyinclude one or more enzymes. Enzymes can be classified on the basis ofthe handbook Enzyme Nomenclature from NC-IUBMB, 1992), see also theENZYME site at the internet: http://www.expasy.ch/enzyme/. ENZYME is arepository of information relative to the nomenclature of enzymes. It isprimarily based on the recommendations of the Nomenclature Committee ofthe International Union of Biochemistry and Molecular Biology (IUB-MB),Academic Press, Inc., 1992, and it describes each type of characterizedenzyme for which an EC (Enzyme Commission) number has been provided(Bairoch, The ENZYME database, 2000, Nucleic Acids Res. 28:304-305).This IUB-MB Enzyme nomenclature is based on their substrate specificityand occasionally on their molecular mechanism; such a classificationdoes not reflect the structural features of these enzymes.

Another classification of certain glycoside hydrolase enzymes, such asendoglucanase, xylanase, galactanase, mannanase, dextranase andalpha-galactosidase, in families based on amino acid sequencesimilarities has been proposed a few years ago. They currently fall into90 different families: See the CAZy(ModO) internet site (Coutinho, P. M.& Henrissat, B. (1999) Carbohydrate-Active Enzymes server at URL:http://afmb.cnrs-mrs.fr/˜cazy/CAZY/index.html (corresponding papers:Coutinho, P. M. & Henrissat, B. (1999) Carbohydrate-active enzymes: anintegrated database approach. In “Recent Advances in CarbohydrateBioengineering”, H. J. Gilbert, G. Davies, B. Henrissat and B. Svenssoneds., The Royal Society of Chemistry, Cambridge, pp. 3-12; Coutinho, P.M. & Henrissat, B. (1999) The modular structure of cellulases and othercarbohydrate-active enzymes: an integrated database approach. In“Genetics, Biochemistry and Ecology of Cellulose Degradation”, K.Ohmiya, K. Hayashi, K. Sakka, Y. Kobayashi, S. Karita and T. Kimuraeds., Uni Publishers Co., Tokyo, pp. 15-23).

Thus the composition of the invention may also comprise at least oneother enzyme selected from the group comprising of phytase (EC 3.1.3.8or 3.1.3.26); xylanase (EC 3.2.1.8); galactanase (EC 3.2.1.89);alpha-galactosidase (EC 3.2.1.22); protease (EC 3.4); phospholipase A1(EC 3.1.1.32); phospholipase A2 (EC 3.1.1.4); lysophospholipase (EC3.1.1.5); phospholipase C (3.1.4.3); phospholipase D (EC 3.1.4.4);amylase such as, for example, alpha-amylase (EC 3.2.1.1); lysozyme (EC3.2.1.17); and beta-glucanase (EC 3.2.1.4 or EC 3.2.1.6), or any mixturethereof.

In a particular embodiment, the composition of the invention comprises aphytase (EC 3.1.3.8 or 3.1.3.26). Examples of commercially availablephytases include Bio-Feed™ Phytase (Novozymes), Ronozyme® P and HiPhos™(DSM Nutritional Products), Natuphos™ (BASF), Finase® and Quantum® Blue(AB Enzymes), the Phyzyme® XP (Verenium/DuPont) and Axtra® PHY (DuPont).Other preferred phytases include those described in, e.g., WO 98/28408,WO 00/43503, and WO 03/066847.

In a particular embodiment, the composition of the invention comprises axylanase (EC 3.2.1.8). Examples of commercially available xylanasesinclude Ronozyme® WX and G2 (DSM Nutritional Products), Econase® XT andBarley (AB Vista), Xylathin® (Verenium) and Axtra® XB(Xylanase/beta-glucanase, DuPont)

In a particular embodiment, the composition of the invention comprises aprotease (EC 3.4). Examples of commercially available proteases includeRonozyme® ProAct (DSM Nutritional Products).

Manufacturing

The composition of the invention can, e.g., be manufactured as mashcomposition (non-pelleted) or pelleted composition. The bacteriacultures and optionally enzymes can be added as solid or liquidformulations. For example, for mash composition a solid or liquidculture formulation may be added before or during the ingredient mixingstep. Typically a liquid culture preparation comprises the culture ofthe invention optionally with a polyol, such as glycerol, ethyleneglycol or propylene glycol, and is added after the pelleting step, suchas by spraying the liquid formulation onto the pellets.

The enzyme may be added to the composition as a granule, which isoptionally pelleted or extruded. The granule typically comprises a coreparticle and one or more coatings, which typically are salt and/or waxcoatings. The core particle can either be a homogeneous blend of anactive compound optionally together with salts (e.g., organic orinorganic zinc or calcium salt) or an inert particle with an activecompound applied onto it. The active compound is the culture of theinvention optionally combined with one or more enzymes. The inertparticle may be water soluble or water insoluble, e.g., starch, a sugar(such as sucrose or lactose), or a salt (such as NaCl, Na₂SO₄). The saltcoating is typically at least 1 μm thick and can either be oneparticular salt or a mixture of salts, such as Na₂SO₄, K₂SO₄, MgSO₄and/or sodium citrate. Other examples are those described in, e.g., WO2008/017659, WO 2006/034710, WO 97/05245, WO 98/54980, WO 98/55599, WO00/70034 or polymer coating such as described in WO 01/00042.

Preferred Embodiments

Preferred embodiments of the invention are described in the two set ofitems herein below.

Item Set I:

1. A Bacillus strain characterized in that:

i) the Bacillus strain is non-hemolytic,

ii) the Bacillus strain has antimicrobial activity against Clostridiumperfringens and

iii) the Bacillus strain improves body weight gain and/or feedconversion rate in chickens fed with the Bacillus strain.

2. A Bacillus strain characterized in that:

i) the Bacillus strain is non-hemolytic,

ii) the Bacillus strain has antimicrobial activity against Clostridiumperfringens and

iii) the Bacillus strain is sensitive to at least seven such as eight ofthe antibiotics selected from the group consisting of Vancomycin,Clindamycin, Chloramphenicol, Gentamicin, Kanamycin, Streptomycin,Erythromycin and Tetracycline.

3. A Bacillus strain characterized in that:

i) the Bacillus strain is non-hemolytic,

ii) the Bacillus strain is sensitive to at least seven such as eight ofthe antibiotics selected from the group consisting of Vancomycin,Clindamycin, Chloramphenicol, Gentamicin, Kanamycin, Streptomycin,Erythromycin and Tetracycline and

iii) the Bacillus strain improves body weight gain and/or feedconversion rate in chickens fed with the Bacillus strain.

4. A Bacillus strain characterized in that:

i) the Bacillus strain is non-hemolytic,

ii) the Bacillus strain has antimicrobial activity against Clostridiumperfringens and optionally

iii) the Bacillus strain has enzymatic activity under aerobic and/oranaerobic conditions that hydrolyzes one or more of the substratesselected from the group consisting of Amylose, Arabinan, Arabinoxylan,Casein and Xylan.

5. The Bacillus strain according to any of items 1 to 3, wherein theBacillus strain has enzymatic activity under aerobic and/or anaerobicconditions that hydrolyzes one or more of the substrates selected fromthe group consisting of Amylose, Arabinan, Arabinoxylan, Casein andXylan (e.g., determined as described in Example 6).6. The Bacillus strain according to any of items 2 to 4, wherein theBacillus strain improves one or more performance parameters in poultryselected from the list consisting of body weight gain, EuropeanProduction Efficacy Factor and feed conversion rate in poultry fed withthe Bacillus strain.7. The Bacillus strain according to item 3, wherein the Bacillus strainhas antimicrobial activity against Clostridium perfringens (e.g.,determined as described in Example 2).8. The Bacillus strain according to any of items 1 and 4, wherein theBacillus strain is sensitive to at least seven such as eight of theantibiotics selected from the group consisting of Vancomycin,Clindamycin, Chloramphenicol, Gentamicin, Kanamycin, Streptomycin,Erythromycin and Tetracycline (e.g., determined as described in Example5).9. The Bacillus strain according to any of the previous items, whereinthe Bacillus strain comprises 16S rDNA that is more than 98% sequenceidentity to SEQ ID NO: 1 and/or more than 98% sequence identity to SEQID NO: 2 and/or more than 98% sequence identity to SEQ ID NO: 3 and/ormore than 98% sequence identity to SEQ ID NO: 4.10. The Bacillus strain according to any of the previous items, whereinthe Bacillus strain is a Bacillus subtilis strain or a Bacillusamyloliquefaciens strain.11. The Bacillus strain according to any of the previous items, whereinthe Bacillus strain has antimicrobial effect against E. coli (e.g.,determined as described in Example 4).12. The Bacillus strain according to any of the previous items, whereinthe Bacillus strain is the Bacillus strain having deposit accessionnumber DSM 29869, or a strain having all of the identifyingcharacteristics of Bacillus DSM 29869 or a mutant thereof that hasantimicrobial activity against Clostridium perfringens.13. The Bacillus strain according to any of the previous items, whereinthe Bacillus strain is the Bacillus strain having deposit accessionnumber DSM 29870, or a strain having all of the identifyingcharacteristics of Bacillus DSM 29870 or a mutant thereof that hasantimicrobial activity against Clostridium perfringens.14. The Bacillus strain according to any of the previous items, whereinthe Bacillus strain is the Bacillus strain having deposit accessionnumber DSM 29871, or a strain having all of the identifyingcharacteristics of Bacillus DSM 29871 or a mutant thereof that hasantimicrobial activity against Clostridium perfringens.15. The Bacillus strain according to any of the previous items, whereinthe Bacillus strain is the Bacillus strain having deposit accessionnumber DSM 29872, or a strain having all of the identifyingcharacteristics of Bacillus DSM 29872 or a mutant thereof that hasantimicrobial activity against Clostridium perfringens.16. A Bacillus having deposit accession number DSM 29869 or a strainhaving all of the identifying characteristics of Bacillus DSM 29869 or amutant thereof.17. A Bacillus having deposit accession number DSM 29870 or a strainhaving all of the identifying characteristics of Bacillus DSM 29870 or amutant thereof.18. A Bacillus having deposit accession number DSM 29871 or a strainhaving all of the identifying characteristics of Bacillus DSM 29871 or amutant thereof.19. A Bacillus having deposit accession number DSM 29872 or a strainhaving all of the identifying characteristics of Bacillus DSM 29872 or amutant thereof.20. The Bacillus strain according to any of the previous items, whereinthe Bacillus strain has a high compatibility with monensin such as beingcompatible with at least 2.3 μg/ml monensin as determined in Example 12(such as at least 2.4 μg/ml monensin, such as at least 2.5 μg/mlmonensin, such as at least 2.6 μg/ml monensin or such as at least 2.7μg/ml monensin as determined in Example 12).21. A composition comprising spores of the Bacillus strain according toany of items 1-20.22. The composition according to item 21, wherein the Bacillus spores ofthe composition are present as dried spores.23. The composition according to any of items 21 to 22 which furthercomprises a carrier.24. The composition according to item 23, wherein the carrier comprisesone or more of the following compounds: water, glycerol, ethyleneglycol, 1, 2-propylene glycol or 1, 3-propylene glycol, sodium aluminiumsilicate, sodium chloride, sodium benzoate, potassium sorbate, sodiumsulfate, potassium sulfate, magnesium sulfate, sodium thiosulfate,calcium carbonate, sodium citrate, dextrin, maltodextrin, glucose,sucrose, sorbitol, lactose, wheat flour, wheat bran, corn gluten meal,starch, farigel, cassava cores, sodium aluminium silicate, colloidalamorphous silica, Sipernat 50S, polyethylene glycol 200, polyethyleneglycol 400, polyethylene glycol 600, polyethylene glycol 1000,polyethylene glycol 1500, polyethylene glycol 4000, carbopol andcellulose.25. The composition according to any of items 21 to 24, wherein thecomposition comprises from 10⁵ to 10¹⁸ CFU/g of isolated Bacillusspores.26. The composition according to any of items 21 to 25 which furthercomprises one or more components selected from the list consisting of:

one or more enzymes;

one or more additional microbes;

one or more vitamins;

one or more minerals;

one or more amino acids; and

one or more other feed ingredients.

27. The composition according to any of items 21 to 26, wherein thebacterial count of each Bacillus spore is between 1×10⁴ and 1×10¹⁸CFU/kg of composition, preferably between 1×10⁷ and 1×10¹⁶ CFU/kg ofcomposition, more preferably between 1×10¹⁰ and 1×10¹⁵ CFU/kg ofcomposition and most preferably between 1×10¹¹ and 1×10¹⁴ CFU/kg ofcomposition.28. The composition according to any of items 21 to 26, wherein thebacterial count of each Bacillus spore is between 1×10⁴ and 1×10¹⁸CFU/kg of composition, preferably between 1×10⁶ and 1×10¹⁵ CFU/kg ofcomposition, and more preferably between 1×10⁷ and 1×10¹¹ CFU/kg ofcomposition.29. A biologically pure culture of the Bacillus strain according to anyof items 1 to 20.30. An isolated Bacillus strain according to any of items 1 to 20.31. An isolated Bacillus strain selected from the group consisting ofBacillus strain DSM 29869, DSM 29870, DSM 29871 and DSM 29872, or astrain having all of the identifying characteristics of Bacillus DSM29869, DSM 29870, DSM 29871 and DSM 29872 or a mutant thereof.32. The isolated Bacillus strain according to item 31, wherein theidentifying characteristics can one or more (such as all) of thecharacteristics selected from the group consisting of

i) non-hemolytic, e.g., as determined in Example 1,

ii) antimicrobial activity against Clostridium perfringens, e.g., asdetermined in Example 2,

iii) antimicrobial activity against E. coli, e.g., as determined inExample 4,

iv) sensitive to Vancomycin, Clindamycin, Chloramphenicol, Gentamicin,Kanamycin, Streptomycin, Erythromycin and Tetracyclin, and

v) high compatibility with monensin such as being compatible with atleast 2.3 μg/ml monensin as determined in Example 12 (such as at least2.4 μg/ml monensin, such as at least 2.5 μg/ml monensin, such as atleast 2.6 μg/ml monensin or such as at least 2.7 μg/ml monensin asdetermined in Example 12).

Item Set II:

1. A Bacillus strain characterized in:

i) having deposit accession number DSM 29870 or a strain having all ofthe identifying characteristics of Bacillus DSM 29870 or a mutantthereof;

ii) having deposit accession number DSM 29869 or a strain having all ofthe identifying characteristics of Bacillus DSM 29869 or a mutantthereof;

iii) having deposit accession number DSM 29871 or a strain having all ofthe identifying characteristics of Bacillus DSM 29871 or a mutantthereof; or

iv) having deposit accession number DSM 29872 or a strain having all ofthe identifying characteristics of Bacillus DSM 29872 or a mutantthereof.

2. The Bacillus strain according to item 1, wherein the Bacillus strainis non-hemolytic.

3. The Bacillus strain according to any of the previous items, whereinthe Bacillus strain has antimicrobial activity against Clostridiumperfringens (e.g., determined as described in Example 2).

4. The Bacillus strain according to any of the previous items, whereinthe Bacillus strain improves body weight gain and/or feed conversionrate in chickens fed with the Bacillus strain.

5. The Bacillus strain according to any of the previous items, whereinthe Bacillus strain comprises 16S rDNA that is more than 98% sequenceidentity to SEQ ID NO: 1 and/or more than 98% sequence identity to SEQID NO: 2 and/or more than 98% sequence identity to SEQ ID NO: 3 and/ormore than 98% sequence identity to SEQ ID NO: 4.6. The Bacillus strain according to any of the previous items, whereinthe Bacillus strain comprises 16S rDNA that is more than 99% sequenceidentity to SEQ ID NO: 1 and/or more than 99% sequence identity to SEQID NO: 2 and/or more than 99% sequence identity to SEQ ID NO: 3 and/ormore than 99% sequence identity to SEQ ID NO: 4.7. The Bacillus strain according to any of the previous items, whereinthe Bacillus strain is a Bacillus subtilis strain or a Bacillusamyloliquefaciens strain.8. The Bacillus strain according to any of the previous items, whereinthe Bacillus strain has antimicrobial effect against E. coli (e.g.,determined as described in Example 4).9. The Bacillus strain according to any of the previous items, whereinthe Bacillus strain is an isolated Bacillus strain.10. A biologically pure culture of the Bacillus strain according to anyof items 1 to 9.11. The Bacillus strain according to any of the previous items, whereinthe Bacillus strain is sensitive to at least seven such as eight of theantibiotics selected from the group consisting of Vancomycin,Clindamycin, Chloramphenicol, Gentamicin, Kanamycin, Streptomycin,Erythromycin and Tetracycline (e.g., determined as described in Example5).12. The Bacillus strain according to any of the previous items, whereinthe Bacillus strain has enzymatic activity under aerobic and/oranaerobic conditions that hydrolyzes one or more of the substratesselected from the group consisting of Amylose, Arabinan, Arabinoxylan,Casein and Xylan (e.g., determined as described in Example 6).13. The Bacillus strain according to any of the previous items, whereinthe Bacillus strain improves one or more performance parameters inpoultry selected from the list consisting of body weight gain, EuropeanProduction Efficacy Factor and feed conversion rate in poultry fed withthe Bacillus strain.14. The Bacillus strain according to any of the previous items, whereinthe Bacillus strain has a high compatibility with monensin such as beingcompatible with at least 2.3 μg/ml monensin as determined in Example 12(such as at least 2.4 μg/ml monensin, such as at least 2.5 μg/mlmonensin, such as at least 2.6 μg/ml monensin or such as at least 2.7μg/ml monensin as determined in Example 12).15. A composition comprising spores of the Bacillus strain according toany of items 1-14.16. The composition according to item 15, wherein the Bacillus spores ofthe composition are present as spores such as dried spores such as spraydried spores.17. The composition according to item 16, wherein the bacterial count ofeach Bacillus spore is between 1×10⁴ and 1×10¹⁸ CFU/kg of composition,preferably between 1×10⁶ and 1×10¹⁵ CFU/kg of composition, and morepreferably between 1×10⁷ and 1×10¹¹ CFU/kg of composition.18. The composition according to any of items 15 to 17 which furthercomprises a carrier.19. The composition according to item 18, wherein the carrier comprisesone or more of the following compounds: water, glycerol, ethyleneglycol, 1, 2-propylene glycol or 1, 3-propylene glycol, sodium aluminiumsilicate, sodium chloride, sodium benzoate, potassium sorbate, sodiumsulfate, potassium sulfate, magnesium sulfate, sodium thiosulfate,calcium carbonate, sodium citrate, dextrin, maltodextrin, glucose,sucrose, sorbitol, lactose, wheat flour, wheat bran, corn gluten meal,starch, farigel, cassava cores, sodium aluminium silicate, colloidalamorphous silica, Sipernat 50S, polyethylene glycol 200, polyethyleneglycol 400, polyethylene glycol 600, polyethylene glycol 1000,polyethylene glycol 1500, polyethylene glycol 4000, carbopol. andcellulose.20. The composition according to item 18 or 19, wherein the carriercomprises calcium carbonate.21. The composition according to any of items 15 to 20, wherein thecomposition comprises from 10⁵ to 10¹⁸ CFU/g of isolated Bacillusspores.22. The composition according to any of items 15 to 21 which furthercomprises one or more components selected from the list consisting of:

one or more enzymes;

one or more additional microbes;

one or more vitamins;

one or more minerals;

one or more amino acids; and

one or more other feed ingredients.

23. The composition according to any of items 15 to 22, wherein thebacterial count of each Bacillus spore is between 1×10⁴ and 1×10¹⁸CFU/kg of composition, preferably between 1×10⁷ and 1×10¹⁶ CFU/kg ofcomposition, more preferably between 1×10¹⁰ and 1×10¹⁵ CFU/kg ofcomposition and most preferably between 1×10¹¹ and 1×10¹⁴ CFU/kg ofcomposition.24. The composition according to any of items 15 to 23, wherein thebacterial count of each Bacillus spore is between 1×10⁴ and 1×10¹⁸CFU/kg of composition, preferably between 1×10⁶ and 1×10¹⁵ CFU/kg ofcomposition, and more preferably between 1×10⁷ and 1×10¹¹ CFU/kg ofcomposition.25. An isolated Bacillus strain selected from the group consisting ofBacillus strain DSM 29869, DSM 29870, DSM 29871 and DSM 29872, or astrain having all of the identifying characteristics of Bacillus DSM29869, DSM 29870, DSM 29871 and DSM 29872 or a mutant thereof.26. The isolated Bacillus strain according to item 25, wherein theidentifying characteristics can one or more (such as all) of thecharacteristics selected from the group consisting of

i) non-hemolytic, e.g., as determined in Example 1,

ii) antimicrobial activity against Clostridium perfringens, e.g., asdetermined in Example 2,

iii) antimicrobial activity against E. coli, e.g., as determined inExample 4,

iv) sensitive to Vancomycin, Clindamycin, Chloramphenicol, Gentamicin,Kanamycin, Streptomycin, Erythromycin and Tetracyclin, and

v) high compatibility with monensin such as being compatible with atleast 2.3 μg/ml monensin as determined in Example 12 (such as at least2.4 μg/ml monensin, such as at least 2.5 μg/ml monensin, such as atleast 2.6 μg/ml monensin or such as at least 2.7 μg/ml monensin asdetermined in Example 12).

27. The isolated Bacillus strain according to item 25 or 26 for use oftreatment of necrotic enteritis and/or a Clostridium perfringensinfection.

28. An isolated Bacillus strain selected from the group consisting ofBacillus strain DSM 29869, DSM 29871 and DSM 29872, or a strain havingall of the identifying characteristics of Bacillus DSM 29869, DSM 29871and DSM 29872 or a mutant thereof for treatment of necrotic enteritisand/or a Clostridium perfringens infection.29. Use of the Bacillus strain or the composition according to any ofthe previous items to improve one or more performance parameters of ananimal selected from the group consisting of: improving the feedconversion ratio, improving the body weight gain, improving the EuropeanProduction Efficacy Factor, improving the feed efficiency and improvingthe health, e.g., of poultry such as chickens.30. The use according to item 29, wherein the Bacillus strain isselected from the group consisting of Bacillus strain DSM 29869, DSM29871 and DSM 29872, or a strain having all of the identifyingcharacteristics of Bacillus DSM 29869, DSM 29871 and DSM 29872 or amutant thereof.31. A method of improving one or more performance parameters of ananimal selected from the group consisting of: improving the feedconversion ratio, improving the body weight gain, improving the EuropeanProduction Efficacy Factor, improving the feed efficiency and improvingthe health, comprising administering the Bacillus strain or thecomposition according to any of the previous items to the animal.32. The method according to item 31, wherein the Bacillus strain isselected from the group consisting of Bacillus strain DSM 29869, DSM29871 and DSM 29872, or a strain having all of the identifyingcharacteristics of Bacillus DSM 29869, DSM 29871 and DSM 29872 or amutant thereof.33. A method for feeding an animal comprising administering the Bacillusstrain or the composition according to any of the previous items to saidanimal optionally in conjunction with other animal feed ingredients.34. The method for feeding an animal according to item 33 wherein theanimal is selected from the group consisting of poultry and swine.

EXAMPLES Example 1: Screening for Hemolysis Negative Bacillus Strains

Hemolysis was tested according to the Technical Guidance on theassessment of the toxigenic potential of Bacillus species used in animalnutrition, EFSA Journal 2011; 9(11):2445.

Sheep blood agar plates were purchased as ready to use (Becton Dickensonart 254053 or 254087). Alternatively, the agar plates can be prepared byadding 5% defibrinated sheep blood (obtained from Statens SerumInstitute, Denmark) to TS-agar (Oxoid CM 131). Agar should be autoclavedat 121° C. for 20 minutes and cooled down to about 40° C. before addingthe blood immediately before pouring the plates.

The Bacillus strains were taken from the preservation at −80° C. andstreaked on TS-agar plate, which was incubated at 30° C. overnight oruntil growth appeared. From a single colony as little as possible of thematerial was used to streak a line on ¼ of an agar plate. The plate wasincubated at 30° C. for 72 hours. Hemolysis/clearing zones of theBacillus strains to be tested were compared with the positive andnegative control. As positive control Bacillus subtilis ATCC21332 wasused. As negative control Bacillus subtilis 168, (BGSC-1A1, BacillusGenetic Stock Center) was used.

In a screening of 599 independent isolates of Bacillus 223 strains (37%)were hemolysis negative. In another screening 21 of 65 independentisolates of Bacillus (32%) were hemolysis negative. Both screeningsexclusively comprised strains that based on identification by 16S rDNAsequencing belong to Bacillus subtilis, Bacillus licheniformis, Bacilluspumilus or Bacillus amyloliquefaciens or close relatives to these fourspecies.

The non-Hemolytic Bacillus strains therefore seem to be common andfairly abundant in nature, but only comprising a minority of the naturalstrains. The non-hemolytic strains seem to be more abundant in Bacilluslicheniformis, while most Bacillus amyloliquefaciens strains appearhemolytic. All the non-hemolytic strains were tested for inhibition ofgrowth of Clostridium perfringens. The following strains from screeningwere all hemolysis negative and among those selected for furtherstudies: DSM 29869, DSM 29870, DSM 29871 and DSM 29872. Clostat(Bacillus PB6) was determined to be hemolytic.

Example 2: Determination of Inhibition of Growth of Clostridiumperfringens

All the non-hemolytic strains were tested for inhibition of growth ofClostridium perfringens strains 23 and 48 (both are netB positive)[Gholamiandekhordi et al., 2006, Molecular and phenotypicalcharacterization of Clostridium perfringens isolates from poultry flockswith different disease status, Vet. Microbiol. 113:143-152], were grownovernight in tryptic soy broth (BD part 211822) supplemented with 0.6%yeast extract (BD part 212750) at 35° C. under static anaerobicconditions. 250 μL of the overnight culture of Clostridium perfringenswas added to 250 mL of tryptic soy agar supplemented with 0.6% yeastextract at 40° C. and poured into rectangular petri plates (Nunc part267060). The inoculated agar was allowed to cool at room temperatureafter which an 8 mm diameter well was made in the agar. Plates werestored in absence of oxygen until use.

The Bacillus strain DSM 29869, DSM 29870, DSM 29871, or DSM 29872 wasgrown overnight in tryptic soy broth at 35° C. under aerobic conditions.1000 μL of Bacillus culture was collected and fractionated intocell-free supernatant and cells by centrifugation. 20 μL of cell-freesupernatant or 100× diluted cells in phosphate buffered saline wereadded directly to the wells in the Clostridium perfringens inoculatedagar plates. A control well contained 20 μL of phosphate buffer saline.The plates were incubated for 18 hours at 35° C. under anaerobicconditions.

Inhibition of the Clostridium perfringens strain was noted by a circularclearing zone around the well of interest. The phosphate buffer salinewell was considered a negative control based on lack of clearing zonearound the well.

Cell-free supernatant and 100× diluted cells of Bacillus strains DSM29869, DSM 29870, DSM 29871, and DSM 29872 were able to consistentlyinhibit growth of C. perfringens strains 23 and 48 in vitro. Inhibitionwas also seen by competitor strain “CloSTAT”, for both supernatant andcells, but was not seen with competitor strain GalliproTect. “CloSTAT”is a strain of Bacillus amyloliquefaciens that was isolated from thecommercial DFM product CloSTAT, Kemin. “GalliproTect” is a strain ofBacillus licheniformis that was isolated from the commercial productGallipro Tect, Chr Hansen.

Example 3: Identification, Characterization and Deposit of theBiological Material

The following biological materials were deposited under the terms of theBudapest Treaty at Leibniz-Institut DSMZ-Deutsche Sammlung vonMikro-organismen and Zellkulturen GmbH, Inhoffenstraße 7 B, 38124Braunschweig Germany, and given the following accession numbers:

TABLE 3.1 Deposit of Biological Material Identification Accession NumberDate of Deposit Bacillus amyloliquefaciens DSM 29869 Jan. 12, 2015Bacillus subtilis DSM 29870 Jan. 12, 2015 Bacillus subtilis DSM 29871Jan. 12, 2015 Bacillus amyloliquefaciens DSM 29872 Jan. 12, 2015

The strains have been deposited under conditions that assure that accessto the culture will be available during the pendency of this patentapplication to one determined by foreign patent laws to be entitledthereto. The deposits represent a substantially pure culture of thedeposited strain. The deposits are available as required by foreignpatent laws in countries wherein counterparts of the subject applicationor its progeny are filed. However, it should be understood that theavailability of a deposit does not constitute a license to practice thesubject invention in derogation of patent rights granted by governmentalaction.

Sequencing of 16S rDNA Genes

DNA was extracted from a culture of DSM 29869, DSM 29870, DSM 29871 andDSM 29872 using QiaAmp DNA Blood Mini Kit (Qiagen art 51106). The kitwas used as recommended for extraction of DNA from gram positivebacteria.

16S rDNA was amplified in a total volume of 50 μl by mixing: 10 pmol ofeach of Primer 16S F and 16S R, 0.2 mM of each nucleotide, 2.5 unitsAmpli taq, 1× Ampli taq buffer, 5 μl DNA template and by using thefollowing PCR program: 94° C. 2 min (94° C. 30 s, 52° C. 30 S, 72° C. 1min)×35, 72° C. 10 min on a Perkin Elmer PCR machine. The PCR productwas sequenced by Novozymes DNA sequencing facility using primer 530R,357F, 1390R and 1100F.

TABLE 3.2 Primers: Primer Sequence SEQ ID NO  16S-F5′-GAGTTTGATCCTGGCTCAG-3′ SEQ ID NO: 8  16S-R 5′-AGAAAGGAGGTGATCCAGCC-3′SEQ ID NO: 9  794-R 5′-ATCTAATCCTGTTTGCTCCCC-3′ SEQ ID NO: 10  357-F5′-TACGGGAGGCAGCAG-3′ SEQ ID NO: 11 1390-R 5′-CGGTGTGTRCAAGGCCC-3′SEQ ID NO: 12 1000-F 5′-CAACGAGCGCAACCCT′, SEQ ID NO: 13

Degeneration of primer 1390-R: R is A or G. The 16 S rDNA sequences fromDSM 29869, DSM 29870, DSM 29871 and DSM 29872 are shown as SEQ ID NO:1-4 in the sequence listing respectively. The 16 S rDNA sequences fromDSM 29869, DSM 29870, DSM 29871 and DSM 29872 were analyzed by BLASTagainst EMBL database and showed identity to 16 S rDNA sequences ofBacillus subtilis (SEQ ID NO: 2 and SEQ ID NO: 3) and to Bacillusamyloliquefaciens (SEQ ID NO: 1 and SEQ ID NO: 4).

In order to study the phylogenetic affiliation of SEQ ID NO: 1 to SEQ IDNO: 4 the sequences were analyzed by a ClustalW alignment in MegAlign(DNASTAR) using SEQ ID NO: to SEQ ID NO: 7 as benchmark. These sequencesare reference 16S rDNA sequences of the type strains of Bacillusvallismortis taken from AB021198 (SEQ ID NO: 5), Bacillus subtilis takenfrom AJ276351 (SEQ ID NO: 6) and Bacillus amyloliquefaciens taken fromAB255669 (SEQ ID NO: 7).

The ClustalW alignment of SEQ ID NO: 1 to SEQ ID NO: 7 (FIG. 1) shows 7nucleotide positions where 2 or more sequences have a nucleotide thatdeviates from the other. For numbering the positions in SEQ ID NO: 6 areused. At position 152 SEQ ID NO: 2 is identical to Bacillusamyloliquefaciens and Bacillus vallismortis, but different from the restthat are identical to each other. At position 174 SEQ ID NO: 1 and SEQID NO: 4 are identical to Bacillus amyloliquefaciens, but different fromthe rest that are identical to each other. At position 257 SEQ ID NO: 4is identical to Bacillus amyloliquefaciens, but different from the restthat are identical to each other. At position 437, 444 and 455 SEQ IDNO: 2 and SEQ ID NO: 3 are identical to Bacillus subtilis, but differentfrom the rest that are identical to each other. At position 1223 SEQ IDNO: 1 is identical to Bacillus amyloliquefaciens, but different from therest that are identical to each other. The variation in the 16S rDNAgenes support the species affiliation seen in the BLAST report.

Description of the Biological Material

Bacillus amyloliquefaciens DSM 29869 was isolated by Novozymes (NovoNordisk) from an environmental sample collected at Jamaica in 1990. Thestrain was identified as Bacillus amyloliquefaciens based on 16S rDNAsequencing.

Bacillus subtilis DSM 29870 was isolated by Novozymes (Novo Nordisk)from an environmental sample collected at Jamaica in 1990. The strainwas identified as Bacillus subtilis based on 16S rDNA sequencing.

Bacillus subtilis DSM 29871 was isolated for Novozymes by a Danishhigh-school student at Brønderslev Gymnasium from a soil sample fromprivate property in Denmark in 2008. The strain was identified asBacillus subtilis based on 16S rDNA sequencing.

Bacillus amyloliquefaciens DSM 29872 was isolated from an environmentalsample from the USA in 2008. The strain was identified as Bacillusamyloliquefaciens based on 16S rDNA sequencing.

Example 4: Determination of Inhibition of Growth of Escherichia coli

Escherichia coli strains, ATCC10536 or ATCC25922, were grown overnightin tryptic soy broth (BD part 211822) supplemented with 0.6% yeastextract (BD part 212750) at 35° C. under static anaerobic conditions.100 μL of the overnight culture of Escherichia coli was added to 250 mLof tryptic soy agar supplemented with 0.6% yeast extract at 40° C. andpoured into rectangular petri plates (Nunc part 267060). The inoculatedagar was then allowed to cool at room temperature after which an 8 mmdiameter well was made in the agar.

The Bacillus strain DSM 29869, DSM 29870, DSM 29871, or DSM 29872 wasgrown overnight in tryptic soy broth at 35° C. under aerobic conditions.1000 μL of Bacillus subtilis culture was collected and fractionated intocell-free supernatant and cells by centrifugation. 20 μL of cell-freesupernatant or 100× diluted cells in phosphate buffer saline were addeddirectly to the wells in the Escherichia coli inoculated agar plates. Acontrol well contained 20 μL of phosphate buffer saline. The plates wereincubated for 18 hours at 30° C. under aerobic conditions.

Inhibition of the Escherichia coli strain was noted by a circularclearing zone around the well of interest. The phosphate buffer salinewell was considered a negative control based on lack of clearing zonearound the well.

Cell-free supernatant and 100× diluted cells of Bacillus strains DSM29869, DSM 29870, DSM 29871, and DSM 29872 were able to consistentlyinhibit growth of the E. coli strains ATCC10535 and ATCC25922 in vitro.Inhibition was also seen by competitor strain CloSTAT, for bothsupernatant and cells.

Example 5: Sensitivity to Antibiotics

The minimal inhibitory concentrations (MIC) of eight antibiotics againstBacillus strains DSM 29869, DSM 29870, DSM 29871, and DSM 29872 weredetermined using broth micro dilution essentially as described in theCLSI guidelines (M07-A9 Methods for Dilution AntimicrobialSusceptibility Tests for Bacteria That Grow Aerobically; 2012). Onlymodification was that volumes were changed; 90 μl Mueller Hinton Broth 2with bacteria was added to 10 μl antibiotics dilutions, cfu's ofbacteria and concentration of antibiotics were changed so finalconcentrations and final cfu's matched the guideline. The plates wereincubated for 20-24 h instead of 16-20 h. The control strain recommendedin the CLSI standard Staphylococcus aureus ATCC 29213 was used ascontrol strain.

Strains:

Bacillus amyloliquefaciens DSM 29869

Bacillus subtilis DSM 29870

Bacillus subtilis DSM 29871

Bacillus amyloliquefaciens DSM 29872

S. aureus ATCC 29213

Antibiotics:

Chloramphenicol (Sigma C1919, 10 mg/ml, solubilized in 96% ethanol)

Clindamycin (Sigma PHR1159, 10 mg/ml, solubilized in water)

Erythromycin (ABBOTICIN from Amdipharm 40501, 50 mg/ml, solubilized in96% ethanol)

Gentamycin (Biomedicals inc., 190057, 10 mg/ml, solubilized in water)

Kanamycin (Sigma, CAS no. 25389-94-0, 50 mg/ml, solubilized in water)

Streptomycin (Sigma, S1277, 10 mg/ml, solubilized in water)

Tetracycline (Sigma, T3383, 10 mg/ml, solubilized in water)

Vancomycin (Sigma, V-8138, 10 mg/ml, solubilized in water)

Mueller Hinton Broth 2 (Sigma/Fluka 90922)

0.9% NaCl (Sigma/RdH 31434/Merck 106404)

Tryptone soya agar plates (Oxoid CM 131)

Microtiter plates: Costar plate, polypropylene, round bottom, Corning3879

Preparation of Bacteria:

A few colonies of Bacillus spp. (<1 day old) were inoculated intoMueller Hinton Broth 2 (MHB) and incubated for around 4 hours at 37° C.OD₆₀₀ (BioPhotometer plus, Eppendorf) was measured and adjusted to 0.25(equivalent to McFarland 0.5) in MHB. For the control strain directcolony suspension was used. A few colonies of S. aureus ATCC 29213 (<1day old) were suspended in MHB and OD₆₀₀ (BioPhotometer plus, Eppendorf)was measured and adjusted to 0.10-0.12 (equivalent to McFarland 0.5) inMHB. The bacterial suspensions were diluted 200× in MHB.

Preparation of Assay Plates:

Antibiotics were diluted to the concentration of 640 μg/mL in MHB. A twofold dilution series was prepared in MHB down to the concentration 0.625μg/ml. 10 μl of each dilution and of each antibiotic was pipetted into amicro-titre plate. Later, when the antibiotics were mixed with thesuspension of bacteria, the samples were diluted 10× (10 μL sample in atotal volume of 100 μl). This resulted in the final test range of0.06-64 μg/ml.

If the plates were not used right away the plates were stored in thefreezer at −20° C. until usage.

90 μl of the bacterial suspensions were added to the assay plates. Theassay plates were incubated in a plastic bag with at wet cloth at 37° C.for 20-24 h. The MIC was determined as the lowest concentration ofantibiotic that completely inhibited growth of bacteria as detected bythe unaided eye.

CFU Estimation:

A 10-fold dilution series in 0.9% NaCl was made to the 10⁻³ of thecultures inoculated into the micro-titre plate. 2× 100 ul from the 10⁻³dilution were plated onto two TSA plates. The plates were incubatedovernight at 37° C. Number of CFU/ml was counted.

Three biological replicates of the assay were performed for Bacillussubtilis DSM 29871, Bacillus amyloliquefaciens DSM 29869 and Bacillussubtilis DSM 29870 (MIC 1-3), while four biological replicates wereperformed for Bacillus amyloliquefaciens DSM 29872 (MIC 2-5).

Results:

The MIC values obtained for B. subtilis DSM 29870 showed that thebreakpoint values were equal to or below the breakpoint values given inthe EFSA guideline (EFSA journal 2012; 10(6):2740). For B. subtilis DSM29869 the MIC values obtained in two out of the three biologicalreplicates showed MIC values equal to or below the breakpoint. In MIC 1the value for Tetracycline was 16. However, due to analytical varianceof the method MIC results of one dilution above the breakpoint is ingeneral accepted and the strain may be regarded as being sensitive.Thus, according to EFSA these two strains are regarded as sensitive toall the 8 antibiotics included in the test (Tables 5.2 and 5.3).

For the strains B. subtilis DSM 29871 and B. amyloliquefaciens DSM 29872the MIC values showed that the two strains were sensitive to seven outof the eight antibiotics (Tables 5.1 and 5.4). An increased tolerancewas observed towards Streptomycin and according to EFSA the strains areclassified as resistant to Streptomycin.

As a control S. aureus ATCC 29213 was tested in parallel and had MICvalues within the ranges given by the CLSI standard (M100-S24Performance Standards for Antimicrobial Susceptibility Testing;informational Supplement, 2014) (Table 5.5).

The amount of bacteria inoculated into the assay plates was measured(CFU/ml). In general the CFU/ml was very close to the target value of5*10⁵ CFU/ml. However, the CFU/ml for Bacillus strains may be associatedwith some uncertainty, since the bacteria tend to aggregate and onceaggregated will only result in one colony forming unit (Tables 5.1 and5.2).

TABLE 5.1 MIC results for B. subtilis DSM 29871 EFSA* MIC 1 MIC 2 MIC 3breakpoints Antibiotic μg/ml μg/ml μg/ml μg/ml Chloramphenicol 8 4 8 8Clindamycin 0.5 0.25 0.5 4 Erythromycin 0.125 0.125 0.125 4 Gentamycin0.25 0.125 0.25 4 Kanamycin 2 1 2 8 Streptomycin 32 16 32 8 Tetracycline2 4 4 8 Vancomycin 0.25 0.25 0.25 4 CFU/ml 3.6*10⁵ n.r. n.r. *EFSAJournal 2012; 10(6): 2740 n.r. not registered

TABLE 5.2 MIC results for B. amyloliquefaciens DSM 29869 EFSA* MIC 1 MIC2 MIC 3 breakpoints Antibiotic μg/ml μg/ml μg/ml μg/ml Chloramphenicol 44 4 8 Clindamycin 1 0.5 1 4 Erythromycin 0.5 0.06 0.06 4 Gentamycin 0.50.125 0.06 4 Kanamycin 2 0.5 0.5 8 Streptomycin 2 4 4 8 Tetracycline 168 8 8 Vancomycin 0.25 0.25 0.25 4 CFU/ml 3.9*10⁵ 2.7*10⁵ 1.2*10⁵ *EFSAJournal 2012; 10(6): 2740

TABLE 5.3 MIC results for B. subtilis DSM 29870 EFSA* MIC 1 MIC 2 MIC 3breakpoints Antibiotic μg/ml μg/ml μg/ml μg/ml Chloramphenicol 4 8 4 8Clindamycin 0.25 0.25 0.25 4 Erythromycin 0.125 0.125 0.125 4 Gentamycin0.25 0.125 0.25 4 Kanamycin 2 1 2 8 Streptomycin 4 4 8 8 Tetracycline0.25 0.25 0.25 8 Vancomycin 0.25 0.25 0.25 4 CFU/ml 4.2*10⁵ 2.1*10⁵1.4*10⁵ *EFSA Journal 2012; 10(6): 2740

TABLE 5.4 MIC results for B. amyloliquefaciens DSM 29872 EFSA* MIC 2 MIC3 MIC 4 MIC 5 breakpoints Antibiotic μg/ml μg/ml μg/ml μg/ml μg/mlChloramphenicol 4 4 4 4 8 Clindamycin 0.5 0.5 0.5 0.5 4 Erythromycin0.06 0.125 0.06 0.06 4 Gentamycin 0.06 0.06 0.125 0.125 4 Kanamycin 10.5 2 1 8 Streptomycin 8 (32) 8 32 >64 8 Tetracycline 0.25 0.25 0.1250.125 8 Vancomycin 0.25 0.25 0.125 0.125 4 CFU/ml 2.7*10⁵ 1.5*10⁵4.7*10⁵ 6.3*10⁵ *EFSA Journal 2012; 10(6): 2740

TABLE 5.5 MIC results for S. aureus ATCC 29213 MIC 1 MIC 2 MIC 3 MIC 4MIC 5 CLSI* breakpoints Antibiotic μg/ml μg/ml μg/ml μg/ml μg/ml μg/mlChloramphenicol 8-16 16 8 16 16    2-16 Clindamycin 0.125 0.25 0.1250.06 0.06   0.06-0.25 Erythromycin 0.5 0.5 0.5 0.5 0.5 0.25-1 Gentamycin0.5 0.5 0.5 1 1 0.12-1 Kanamycin 4 4 4 4 4   1-4 Streptomycin 8 8 8 16 8No information Tetracycline 0.5 1 1 0.5 0.5 0.12-1 Vancomycin 0.5 1 1 11  0.5-2 CFU/ml 5.2*10⁵ 7.0*10⁵ 7.0*10⁵ 7.3*10⁵ 4.3*10⁵ *M100-S24Performance Standards for Antimicrobial Susceptibility Testing;informational Supplement, 2014

Example 6: Enzyme Activity

Preparation of Overnight Pre-Cultures

Overnight cultivation was made in a 96-deep well plate with 1.25 mL ofLB broth (Difco; BD #244610) per well. Each strain was tested induplicate and inoculated with a single colony from a pure StandardMethods Agar plate (SMA plates, Smith River Biologicals #11-00450).Before incubation, a single sterile Aeraseal breathable seal (Fisher#50-212-463) was placed on top of the plate, and the plate was incubatedwith shaking at 35° C. overnight under aerobic conditions.

Determination of Anaerobic Growth

SMA plates were streaked from the overnight cultures using a 10 μLinoculating loop. Up to four strains were streaked per plate, intoseparate quadrants.

Plates were incubated at 39° C. in an AnaeroJar (Thermo ScientificOxoid, Fisher #OXAG0025A) along with an AnaeroGen sachet (ThermoScientific Oxoid, Fisher #OXAN0025A) to maintain anaerobic conditions(˜0.1% oxygen). Work was performed under aseptic conditions inbiological hood, using sterile materials.

Enzyme Activity Determination Using AZCL-Substrate Agar Plates

Preparation of media with AZCL substrates which can be autoclavedAZCL-cellulose (AZCL-HE-Cellulose, I-AZCEL, Megazyme), arabinoxylan(AZCL-Arabinoxylan, wheat, I-AZWAX, Megazyme), arabinan (AZCL-Arabinan,debranched, I-AZDAR, Megazyme):

Into two (2) 500 mL beakers, 300 mL of 0.1× and 1× LB agar were preparedfor each beaker, with a magnetic stir bars added to each. Whilestirring, 100 mL of LB condition from the beakers were added to three(3) 250 mL Wheaton bottles, thus giving 3 bottles with 0.1×LB and 3bottles with 1×LB. AZCL-cellulose, arabinan, and arabinoxylan were thenadded separately to each of a 1× and a 0.1× bottle with stirring. Atotal of 0.05 grams of substrate is added per 100 mL of media. If morethan 100 mL of media is being used, the amount of substrate added isadjusted by using the 0.05 g/100 mL ratio. The media were autoclaved tosterilize. Note: Only these three AZCL substrates can be autoclavedwithout obvious dye release indicating substrate instability. Afterautoclaving, media is kept at 50° C. until ready to pour plates. The pHwas checked, and if necessary adjusted to pH 7 using 10% HCl or 2N NaOH,maintaining sterility.

Preparation of Media with AZCL Substrates which Can't be Autoclaved

These substrates were: AZCL-amylose (I-AZAMY, Megazyme), AZCL-casein(I-AZCAS, Megazyme), AZCL-xylan (Xylan, birchwood (I-AZXBW, Megazyme)

Into two (2) 1000 mL Wheaton bottles, 400 mL of 0.1×LB and 1×LB wasprepared and a magnetic stir bar was added to each. The media wasautoclaved to sterilize, prior to adding AZCL reagent. Afterautoclaving, media was kept at 50-55° C. until ready to add AZCLsubstrate and pour plates. The pH was checked, and if necessary adjustedto pH 7 using 10% HCl or 2N NaOH, maintaining sterility.

Pouring AZCL Substrate Media Agar Plates

Work was performed under aseptic conditions in biological hood, usingsterile materials. These steps were done relatively quickly, so that theagar did not solidify before transferring into the 96-well plate.

For substrates which could be autoclaved (AZCL-cellulose,AZCL-arabinoxylan, AZCL-arabinan): The warm (˜50° C.), autoclaved liquidagar media was magnetically stirred in order to suspend the insolubleAZCL substrate. An aliquot was transferred to a sterile solution basin(Periodic mixing of the material in the solution basin was necessary tokeep the substrate suspended.) A repeating, multi-channel pipette (e.g.,Matrix 1250 μL pipet along with the corresponding 1250 μL sterile pipettips) was used to dispense 180 μL into each well of a sterile 96-wellplate. A set of tips would typically last for up to six columns perplate. Once the plate was filled, a sterile lid was added, and it wasallowed to solidify. One plate was made for each substrate(AZCL-cellulose, AZCL-arabinoxylan, AZCL-arabinan) and condition (0.1×and 1×LB).

For the substrates that could not be autoclaved (AZCL-amylose,AZCL-casein, AZCL-xylan): Into a sterile 250 mL beaker with magneticstir-bar was added 100 mL of warm, sterile 0.1× or 1×LB media (no AZCLsubstrate added). With stirring, add 0.05 mg of AZCL substrate, andcontinue stirring until re-suspended. It may help to add the substrateslowly at first. Once substrate was re-suspended, it was poured intosolution basin and added to plates as described above. One plate wasmade for each substrate (AZCL-cellulose, AZCL-arabinoxylan,AZCL-arabinan) and condition (0.1× and 1×LB).

Inoculating the 96-Well Plates, Incubation, and Evaluation

Work was performed under aseptic conditions in biological hood, usingsterile materials. The AZCL substrate agar plates were inoculated byapplying 2 μL of overnight culture that was dispensed on top of theagar. The added liquid should be visible on top of the agar. When movingto the next plate, be sure that there are no drops left at the end ofthe pipet tips to ensure that each well has been inoculated. Repeat forall AZCL substrate plates. Control enzyme dilutions were prepared, eachat 1/100, diluting in sterile phosphate dilution buffer: 10 μL enzymepreparation+990 μL buffer. Control enzyme wells were then inoculatedwith 10 μL of the appropriate enzyme dilution for each substrate

TABLE 6.1 Enzymes and respective substrates Enzyme Substrate AmylaseAmylose Arabinase Arabinan Cellulase Cellulose Protease Casein XylanaseArabinoxylan, Xylan

TABLE 6.2 Enzyme production under aerobic conditions 30° C. Aerobic, 24h Amy- Arabino- Cellu- organism ID lose Arabinan xylan Casein lose XylanDSM 29869 + + + + − + DSM 29870 + + + + − + DSM 29871 + + + + + + DSM29872 + + + + − +

TABLE 6.3 Enzyme production under anaerobic conditions, 24 hours 30° C.Anaerobic, 24 h Amy- Arabino- Cellu- organism ID lose Arabinan xylanCasein lose Xylan DSM 29869 + + + − − + DSM 29870 + + +/− + − + DSM29871 − + + − + + DSM 29872 + + + − − +

TABLE 6.4 Enzyme production under anaerobic conditions, 48 hours 30° C.Anaerobic, 48 h Amy- Arabino- Cellu- organism ID lose Arabinan xylanCasein lose Xylan DSM 29869 + + + + − + DSM 29870 + + +/− + − + DSM29871 − + + − + + DSM 29872 + + + − − +

Example 7: Efficacy of the DSM 29870 Strain in Clostridium perfringensChallenge Conditions

Material and Methods

Three independent battery studies have been conducted to evaluate theinfluence of the strain DSM 29870 on the development of an inducednecrotic enteritis.

In each experiment, a total of 256 one day-old male broiler chickensCobb×Cobb were allocated to Petersime battery cages (8 birds/cage).Cages were used in factorial and completely randomized design with 8cages per treatment (i.e., 64 animals/treatment).

An unmedicated corn/soybean meal-based commercial broiler starter rationwas formulated (Table 7.1). Feed and water were available ad libitumthroughout the trials.

TABLE 7.1 Composition of the basal experimental diet⁴ (starter d1-d28)Starter (0-28) Ingredients Corn, yellow, ground 56.12 Soybean meal (48)37.50 Fat, poultry 3.00 Dicalcium phosphate 1.75 Limestone 0.80 Salt0.30 Vitamin premix¹ 0.25 DL-Methionine 0.20 Trace mineral premix² 0.08Calculated Nutritional Content ME, kcal/kg 3.096 Protein, % 22.30Lysine, % 1.180 Methionine, % 0.530 Met + Cys, %³ 0.890 ¹Vitamin mixprovided the following (per kg of diet): thiamin•mononitrate, 2.4 mg;nicotinic acid, 44 mg; riboflavin, 4.4 mg; D-Ca pantothenate, 12 mg;vitamin B₁₂ (cobalamin), 12.0 μg; pyridoxine•HCL, 4.7 mg; D-biotin, 0.11mg; folic acid, 5.5 mg; menadione sodium bisuffite complex, 3.34 mg;choline chloride, 220 mg; cholecalciferol, 27.5 ug; trans-retinylacetate, 1,892 ug; all-rac α tocopheryl acetate, 11 mg; ethoxyquin, 125mg. ²Trace mineral mix provided the following (per kg of diet):manganese (MnSO₄•H₂O), 60 mg; iron (FeSO₄•7H₂O), 30 mg; zinc (ZnO), 50mg; copper (CuSO₄•5H₂O), 5 mg; iodine (ethylene diamine dihydroiodide),0.15 mg; selenium (NaSeO₃), 0.3 mg. ³Met = methionine; Cys = cysteine.⁴The basal feed will not contain any probiotic/prebiotic feed additives,NSPases, coccidiostats or antibiotic growth promoter.

The 4 experimental groups consisted in (Table 7.2): T1, non-infected andnon-treated animals; T2, C. perfringens infected and non-treatedanimals; T3, C. perfringens infected animals fed with the basal dietcontaining bacitracin (included at 50 ppm, frCom d1 to d28); T4, C.perfringens infected animals fed with the basal diet containing the DSM29870 strain (included at 1.10⁹ CFU/kg of feed in trial 1 and 5.10⁸CFU/kg of feed in trials 2 and 3, from d1 to d28).

TABLE 7.2 Experimental treatments Treatments T1 Non-infected,non-treated T2 Infected, non-treated T3 Infected, bacitracin T4Infected, DSM 29870

On day 14, all birds were orally inoculated with a coccidial inoculumcontaining approximately 5,000 oocysts of Eimeria maxima per bird.

Once daily on d19, d20 and d21, all birds, except T1, were administered(by oral gavage) 1 mL of a fresh broth culture of C. perfringens.

On d21, three birds from each cage were selected, sacrificed, weighed,and examined for the degree of presence of necrotic enteritis lesions.The scoring was based on a 0 to 3 score, with 0 being normal and 3 beingthe most severe.

The live body weight and the feed were recorded at the start (d1) andthe end of the experiment (d28), on a cage basis, to calculateperformance:

-   -   Body Weight Gain (BWG)=(Live Body Weight)_(d28)−(Live Body        Weight)_(d1)    -   Feed Conversion Ratio (FCR)=ratio between feed consumed and        weight gained

Mortality was recorded daily and the FCR was adjusted accordingly.

Necrotic enteritis lesion scores, total mortality and % of dead birdswith necrotic enteritis lesions were also calculated

Data (n=32) were subjected to an ANOVA, with complete randomized blockdesign using the ANOVA procedure of XLSTAT (Addinsoft 1995-2014) toestablish differences between diets. Pen was considered as theexperimental unit. The model included diets (n=4) and block as fixedeffect. Results are reported as least square means. LS means wereassumed to be different at P≤0.05.

Experimental Results

The results obtained on performance, necrotic enteritis lesion scores,mortality and % of dead birds with necrotic enteritis lesions parametersare shown in Tables 7.3.

TABLES 7.3 Effect of dietary supplementation of the strain DSM 29870 onthe negative impact on production parameters due to induced necroticenteritis in three independent trials. Total Dead birds with BWG⁵ Lesionmortality necrotic enteritis (g/bird) FCR⁵ score⁶ (%) lesions (%) Trial1 (d1-d28) (d1-d28) (d21) (d1-d28) (d1-d28) T1¹ 720a 1.666c 0.0a  7.5b 0.0b T2² 549b 2.071a 0.2a 27.5a 17.2a T3³ 633ab 1.813bc 0.2a  5.0b 1.6b T4⁴ 628ab 1.843bc 0.3a  5.0b  3.1b ¹Non-infected, non-treatedgroup fed with the basal diet. ²Infected, non-treated group fed with thebasal diet. ³Infected group fed with the basal diet supplemented withbacitracin (50 ppm). ⁴Infected group fed with the basal dietsupplemented with DSM 29870 (1.10⁹ CFU/kg of feed). ⁵Means from 8replicates of 8 birds per cage for each group. Raw data (n = 32) wereanalyzed using variance analysis with block (n = 8) and treatments (n =4) as fixed effect. ⁶Means from 8 replicates of 3 birds per cage foreach group. Raw data (n = 32) were analyzed using variance analysis withblock (n = 8) and treatments (n = 4) as fixed effect. a,b,cValues withdifferent superscripts are significantly (P < 0.05) different from eachother. Total Dead birds with BWG⁵ Lesion mortality necrotic enteritis(g/bird) FCR⁵ score⁶ (%) lesions (%) Trial 2 (d1-d28) (d1-d28) (d21)(d1-d28) (d1-d28) T1 685a 1.692b 0.0d  2.5c  0.0c T2 435c 2.404a 1.2a32.5a 20.3a T3 665ab 1.759b 0.6b 12.5bc  6.3c T4 654ab 1.768b 0.4bc25.0ab  9.4bc ¹Non-infected, non-treated group fed with the basal diet.²Infected, non-treated group fed with the basal diet. ³Infected groupfed with the basal diet supplemented with bacitracin (50 ppm). ⁴Infectedgroup fed with the basal diet supplemented with DSM 29870 (5.10⁸ CFU/kgof feed). ⁵Means from 8 replicates of 8 birds per cage for each group.Raw data (n = 32) were analyzed using variance analysis with block (n =8) and treatments (n = 4) as fixed effect. ⁶Means from 8 replicates of 3birds per cage for each group. Raw data (n = 32) were analyzed usingvariance analysis with block (n = 8) and treatments (n = 4) as fixedeffect. a,b,cValues with different superscripts are significantly (P <0.05) different from each other. Total Dead birds with BWG⁵ Lesionmortality necrotic enteritis (g/bird) FCR⁵ score⁶ (%) lesions (%) Trial3 (d1-d28) (d1-d28) (d21) (d1-d28) (d1-d28) T1 667a 1.752c 0.0b 10.0a 0.0c T2 521b 1.988a 0.5a 25.0a 15.6a T3 645a 1.786bc 0.6a 15.0a  4.7bcT4 638a 1.851b 0.4a 10.0a  4.7bc ¹Non-infected, non-treated group fedwith the basal diet. ²Infected, non-treated group fed with the basaldiet. ³Infected group fed with the basal diet supplemented withbacitracin (50 ppm). ⁴Infected group fed with the basal dietsupplemented with DSM 29870 (5.10⁸ CFU/kg of feed). ⁵Means from 8replicates of 8 birds per cage for each group. Raw data (n = 32) wereanalyzed using variance analysis with block (n = 8) and treatments (n =4) as fixed effect. ⁶Means from 8 replicates of 3 birds per cage foreach group. Raw data (n = 32) were analyzed using variance analysis withblock (n = 8) and treatments (n = 4) as fixed effect. a,b,cValues withdifferent superscripts are significantly (P < 0.05) different from eachother.

The data obtained from the three challenge studies showed that thestrain DSM 29870 can counteract the negative impact of an inducednecrotic enteritis on the production parameters with a significanteffect in all challenge trials on the FCR and the mortality due tonecrotic enteritis, relative to the positive control group(non-infected, non-treated animals).

Furthermore, there was no significant difference between the DSM 29870fed group and the bacitracin fed group with regards to all parametersmeasured in these trials.

The challenge studies described above showed that the administration ofthe DSM 29870 strain could prevent necrotic enteritis in broilerchickens.

Example 8: First Performance Trial with Broiler Chickens (521)

Material and Methods

A total of 2000 one day-old male broiler chickens Cobb 500 wereindividually weighed (body weight 42 g±1 g) and allocated in floor pens(50 birds/pen). Pens were used in factorial and completely randomizeddesign with 8 pens per treatment (i.e., 400 animals/treatment). The 5experimental treatments consisted in (Table 8.1): T1, negative controlbasal diets; T2, basal diets containing the strain DSM 29870 (includedat 5.10⁸ CFU/kg of feed, from d1 to d35); T3, basal diets containing thestrain DSM 29871 (included at 5.10⁸ CFU/kg of feed, from d1 to d35); T4basal diets containing the strain DSM 29872 (included at 5.10⁸ CFU/kg offeed, from d1 to d35); T5, basal diet containing a marketed probiotic,GalliPro Max (included at the commercial dose, 8.10⁸ CFU/kg of feed,from d1 to d35).

Basal diets consisted in 3 phases feeding program: starter phase (from 1to day 12), grower phase (from 13 to day 28) and finisher phase (from 29to day 35). Every phases were formulated to meet or exceed animalrequirement and agree with standard commercial US corn-soybeanmeal-based broiler diets. The dietary and raw material composition ofthese diets is given in Table 8.2. All experimental diets includephytase (Ronozyme P, 250 FYT/kg) and did not contain any coccidiostat,NSPase or growth promoting substance. Feed and water were available adlibitum throughout the trial.

TABLE 8.1 Experimental treatments Treatments T1 Control T2 T1 + DSM29870 T3 T1 + DSM 29871 T4 T1 + DSM 29872 T5 T1 + GalliPro Max

TABLE 8.2 Composition of the basal experimental diets Starter GrowerFinisher (0-12d) (13-28d) (29-35d) Ingredients % Corn, yellow, grain64.673 66.460 68.491 Soybean meal, dehulled, solvent 29.020 26.66224.677 Ampro 55 (animal by-product 55% 2.500 3.000 3.000 protein)Calcium carbonate 0.886 0.735 0.684 Fat, vegetable 0.883 1.485 1.702Dicalcium phosphate 0.706 0.612 0.500 Salt, plain (NaCl) 0.439 0.4350.436 Methionine MHA 0.358 0.259 0.221 L-Lysine 0.273 0.208 0.145L-Threonine 98.5 0.103 0.000 0.000 Trace Mineral¹ 0.075 0.075 0.075Vitamin premix ² 0.065 0.050 0.050 Ronozyme P-(ct) 0.019 0.019 0.019Calculated Nutritional Content ME (kcal/kg) 3,067 3,130 3,165 Crudeprotein (%) 20.96 20.03 19.16 Dig. Lysine (%) 1.20 1.10 1.00 Dig.Methionine (%) 0.61 0.52 0.48 Dig. TSAA (%) 0.90 0.80 0.75 Dig.Threonine (%) 0.81 0.68 0.65 Calcium (%) 0.90 0.85 0.8 Avail. phosphorus(%) 0.42 0.42 0.4 ¹Vitamin mix will provide the following (per kg ofdiet): thiamin•mononitrate, 2.4 mg; nicotinic acid, 44 mg; riboflavin,4.4 mg; D-Ca pantothenate, 12 mg; vitamin B₁₂ (cobalamin), 12.0 μg;pyridoxine•HCL, 4.7 mg; D-biotin, 0.11 mg; folic acid, 5.5 mg; menadionesodium bisulfite complex, 3.34 mg; choline chloride, 220 mg;cholecalciferol, 27.5 ug; trans-retinyl acetate, 1,892 ug; all-rac αtocopheryl acetate, 11 mg; ethoxyquin, 125 mg. ² Trace mineral mixprovided the following (per kg of diet): manganese (MnSO₄•H₂O), 60 mg;iron(FeSO₄•7H₂O),30 mg; zinc (ZnO), 50 mg; copper (CuSO₄•5H₂O), 5 mg;iodine (ethylene diaminedihydroiodide), 0.15 mg; selenium (NaSeO₃), 0.3mg.

The live body weight and the feed were recorded at the start (d1) andthe end of the experiment (d35), on a pen basis, to calculateperformance:

-   -   Feed Intake (FI)=(Remaining Feed)_(d35)−(Issued Feed)_(d1)    -   Body Weight Gain (BWG)=(Live Body Weight)_(d35)−(Live Body        Weight)_(d1)    -   Feed Conversion Ratio (FCR)=ratio between feed consumed and        weight gained

Mortality was recorded daily and the FCR was adjusted accordingly.

Data (n=40) were subjected to an ANOVA, with complete randomized blockdesign using the ANOVA procedure of XLSTAT (Addinsoft 1995-2014) toestablish differences between diets. Pen was considered as theexperimental unit. The model included diets (n=5) and block as fixedeffect. Results are reported as least square means. LS means wereassumed to be different at P≤0.05.

Similar calculations were realized in the example 9 and 10.

Similar Calculations were Realized in the Example 3 and 4. ExperimentalResults

The results obtained on performance parameters for the wholeexperimental period are shown in Table 8.3.

TABLE 8.3 Effect of dietary supplementation of the strains DSM 29870,DSM 29871 and DSM 29872 on broiler performance GalliPro DSM 29870² DSM29871² DSM 29872² Max³ supplemented supplemented supplementedsupplemented Parameters⁴ Control¹ group group group group FI (g/bird)3245a 3256a 3221a 3263a 3203a Relative to the — +0.3% −0.7% +0.6% −1.3%control BWG (g/bird) 1987ab 2026ab 2002ab 2038a 1973b Relative to the —+2.0% +0.8% +2.6% −0.7% control FCR   1.633a   1.607b   1.609b   1.602b  1.624ab Relative to the — −1.6% −1.5% −2.0% −0.6% control Mortality(%)   3.25a   3.50a   5.25a   4.5a   6.25a ¹Group fed with the basaldiet. ²DSM 29870/29871/29872 supplemented group fed with the basal dietincluding strain DSM 29870/29871/29872 at 5.10⁸ CFU/kg of feed.³GalliPro Max supplemented group fed with the basal diet includingGalliPro Max at 8.10⁸ CFU/kg of feed. ⁴Means from 8 replicates of 50birds per pen for each group. Raw data (n = 24) were analyzed usingvariance analysis with block (n = 8) and treatments (n = 3) as fixedeffect. a,b,cValues with different superscripts are significantly (P <0.05) different from each other.

Feed additives did not significantly affect animal feed intake relativeto the control. Body weight was slightly increased using the NZB strainswhereas GalliPro Max tended to decrease this parameter.

The DSM 29870, DSM 29871 and DSM 29872 strains also improvedsignificantly the feed conversion ratio by 1.6%, 1.5% and 1.9%,respectively, with no significant differences between the groups fedwith NZB strains. GalliPro Max had only a slight and non-significanteffect on the feed conversion ratio.

None of the probiotic tested here had a significant effect on themortality level.

Example 9: Second Performance Trial with Broiler Chickens (522)

Material and Methods

A total of 900 one day-old male broiler chickens Ross 708 wereindividually weighed (body weight 47.1±1.1 g) and allocated in floorpens (15 birds/pens). Pens were used in factorial and completelyrandomized design with 12 pens per treatment (i.e., 180animals/treatment). The 5 experimental treatments consisted in (Table9.1): T1, negative control basal diets; T2, basal diets containing thestrain DSM 29870 (included at 5.10⁸ CFU/kg of feed, from d1 to d35); T3,basal diets containing the strain DSM 29871 (included at 5.10⁸ CFU/kg offeed, from d1 to d35); T4 basal diets containing the strain DSM 29872(included at 5.10⁸ CFU/kg of feed, from d1 to d35); T5, basal dietcontaining a marketed probiotic, GalliPro Max (included at thecommercial dose, 8.10⁸ CFU/kg of feed, from d1 to d35).

Basal diets consisted in 3 phases feeding program: starter phase (from 1to day 14), grower phase (from 15 to day 21) and finisher phase (from 22to day 35). Every phases were formulated to meet or exceed animalrequirement and agree with standard commercial US corn-soybeanmeal-based broiler diets. The dietary and raw material composition ofthese diets is given in Table 9.2. All experimental diets includedvitamin and mineral premixes, but did not contain any coccidiostat,NSPase, phytase or growth promoting substance. Feed and water wereavailable ad libitum throughout the trial.

TABLE 9.1 Experimental treatments Treatments T1 Control T2 T1 + DSM29870 T3 T1 + DSM 29871 T4 T1 + DSM 29872 T5 T1 + GalliPro Max

TABLE 9.2 Composition of the basal experimental diets Starter GrowerFinisher (0-14d) (15-21d) (22-35d) Ingredients % Corn 57.59 59.20 62.30Wheat Flour 1.00 1.00 1.00 Soybean Meal (46% CP) 30.00 28.00 24.50Poultry Meal 5.00 5.00 5.00 Poultry Fat 2.10 2.86 3.52 Limestone (36%Ca) 1.05 1.02 0.98 Dicalcium Phosphate (18% P) 1.76 1.52 1.42 Salt 0.350.32 0.29 Sodium Bicarbonate 0.16 0.14 0.11 L-Lysine HCL (78%) 0.23 0.220.21 DL-Methionine (99%) 0.36 0.33 0.29 L-Threonine (98%) 0.05 0.04 0.03Selenium premix 0.05 0.05 0.05 Vitamin Mix 0.15 0.15 0.15 Mineral Mix0.15 0.15 0.15 Calculated Nutritional Content ME (kcal/kg) 2968 30323096 Crude Protein (%) 23.3 22.4 20.78 Lysine (%) 1.35 1.29 1.18Methionine (%) 0.69 0.65 0.59 Met + Cys (%) 1.05 1.00 0.92 Threonine (%)0.86 0.81 0.75 Calcium (%) 1.03 0.96 0.91 Phosphorus Avail. (%) 0.460.41 0.39 ¹Vitamin mix will provide the following (per kg of diet):vitamin A, 13 227 513 IU; vitamin D3, 3 968 254 IU; vitamin E, 66 138IU; vitamin B₁₂, 40 mg; biotin, 254 mg; menadione, 3 968 mg; thiamine, 3968 mg; riboflavin, 13 228 mg; d-Pantothenic Acid, 22 046 mg;pyridoxine, 7 937 mg; niacin, 110 229 mg; folic acid, 2 205 mg; SeleniumPremix: selenium, 600 ppm + calcium, 36% ²Trace mineral mix provided thefollowing (per kg of diet): calcium, Min: 15.7% and Max: 18.7%;manganese (Mn), 6.0%; zinc (Zn), 6.0%; iron (Fe), 4.0%; copper (Cu),5000 ppm; iodine (I), 1250 ppm; cobalt (Co), 500 ppmExperimental Results

The results obtained on performance parameters for the wholeexperimental period are shown in Table 9.3.

TABLE 9.3 Effect of dietary supplementation of the strains DSM 29870,DSM 29871 and DSM 29872 on broiler performance GalliPro DSM 29870² DSM29871² DSM 29872² Max³ supplemented supplemented supplementedsupplemented Parameters⁴ Control¹ group group group group FI (g/bird)3573a 3542a 3606a 3515a 3541a Relative to the — −0.9% +0.9% −1.0% −0.9%control BWG (g/bird) 2361a 2416b 2401ab 2424b 2398ab Relative to the —+2.3% +1.7% +2.6% +1.5% control FCR   1.513a   1.467b   1.501bc   1.451a  1.477ab Relative to the — −3.6% −0.8% −4.2% −2.4% control Mortality(%)  15.6ab   7.8a  11.7ab  19.4b  10.0a ¹Group fed with the basal diet²DSM 29870/29871/29872 supplemented group fed with the basal dietincluding strain DSM 29870/29871/29872 at 5.10⁸ CFU/kg of feed.³GalliPro Max supplemented group fed with the basal diet includingGalliPro Max at 8.10⁸ CFU/kg of feed. ⁴Means from 12 replicates of 15birds per pen for each group. Raw data (n = 36) were analyzed usingvariance analysis with block (n = 12) and treatments (n = 3) as fixedeffect. a,b,cValues with different superscripts are significantly (P <0.05) different from each other

Also in this experiment, the administration of the strains DSM 29870,DSM 29871, DSM 29872 and GalliPro Max did not significantly reduce thefeed intake, but improved the body weight gain by 2.3%, 1.7%, 2.6% and1.5%, respectively. Therefore, difference relatively to control wassignificant for DSM 29870 and DSM 29872.

The strains DSM 29870 and DSM 29872 improved significantly the feedconversion ratio by 3.6% and 4.2%, respectively. DSM 29871 and GalliProMax also improved (slightly for DSM 29871) the feed conversion ratio inthis experiment, but not significantly.

The strain DSM 29870 allowed a decrease of the mortality level in anumerically higher manner than GalliPro Max.

Example 10: Third Performance Trial with Broiler Chickens (051)

Material and Methods

A total of 1080 one day-old male broiler chickens Ross PM3 wereindividually weighed (body weight 42±3.5 g) and allocated in floor pens(18 birds/pens). Pens were used in factorial and completely randomizeddesign with 12 pens per treatment (i.e., 216 animals/treatment). The 3experimental treatments consisted in (Table 10.1): T1, negative controlbasal diets; T2, basal diets containing the strain DSM 29870 (includedat 5.10⁸ CFU/kg of feed, from d1 to d35); T3, basal diets containing thestrain DSM 29871 (included at 5.10⁸ CFU/kg of feed, from d1 to d35); T4basal diets containing the strain DSM 29872 (included at 5.10⁸ CFU/kg offeed, from d1 to d35); T5, basal diet containing a marketed probiotic,GalliPro Max (included at the commercial dose, 8.10⁸ CFU/kg of feed,from d1 to d35).

Basal diets consisted in 3 phases feeding program: starter phase (from 1to day 21) and grower phase (from 22 to day 35). Every phases wereformulated to meet or exceed animal requirement and agree with standardcommercial EU corn-soybean meal-based broiler diets. The dietary and rawmaterial composition of these diets is given in Table 10.2. Allexperimental diets included vitamin and mineral premixes, but did notcontain any coccidiostat, NSPase, phytase or growth promoting substance.Feed and water were available ad libitum throughout the trial.

TABLE 10.1 Experimental treatments Treatments T1 Control T2 T1 + DSM29870 T3 T1 + DSM 29871 T4 T1 + DSM 29872 T5 T1 + GalliPro Max

TABLE 10.2 Composition of the basal experimental diets Starter Grower(0-21d) (22-35d) Ingredients % Maize 51.68 55.58 Soybean Meal (48% CP)39.03 34.55 Soybean oil 4.27 5.18 DL-Methionine 0.22 0.17 Calciumcarbonate 1.05 1.05 Dicalcium Phosphate 1.78 1.5 Salt 0.37 0.37 Wheatmiddlings 1.00 1.00 Premix¹ 0.6 0.6 Calculated Nutritional Content ME(kcal/kg) 3000 3100 Crude Protein (%) 22.25 20.5 Fat (%) 6.76 7.7Cellulose (%) 3.15 3 Minerals (%) 5.74 5.3 Lysine (%) 1.25 1.13Methionine (%) 0.55 0.48 Met + Cys (%) 0.92 0.83 Threonine (%) 0.87 0.8Calcium (%) 0.93 0.85 Total phosphorus 0.7 0.63 Phosphorus Avail. (%)0.38 0.33 ¹The premix will provide the following (per kg of diet):vitamin A, 12 000 IU; vitamin D3, 3 000 IU;, vitamin E = 300 IU;,vitamin K3, 3 mg; vitamin B, 2 mg; vitamin B2, 8 mg; vitamin B6, 3 mg;vitamin B12, 0.02 mg; folic acid, 1 mg; biotin, 0.2 mg; calciumpantothenate, 15 mg; nicotinic acid, 40 mg; manganese (Mn), 80 mg; zinc(Zn), 60 mg; iodine (I), 1 mg, iron (Fe), 80 mg; copper (Cu), 15 mg;cobalt (Co), 0.4 mg; selenium (Se), 0.2 mg; magnesium (Mg), 5 mg;Etoxyquin, 0.5 mg; BHA, 0.5 mg, citric acid, 5 mg; phosphoric acid, 5mg.Experimental Results

The results obtained on performance parameters for the wholeexperimental period are shown in Table 10.3.

TABLE 10.3 Effect of dietary supplementation of the strains DSM 29870,DSM 29871 and DSM 29872 on broiler performance GalliPro DSM 29870² DSM29871² DSM 29872² Max³ supplemented supplemented supplementedsupplemented Parameters⁴ Control¹ group group group group FI (g/bird)3217a 3295a 3281a 3230a 3246a Relative to the — +2.4% +2.0% +0.4% +0.9%control BWG (g/bird) 2019b 2149a 2167a 2124a 2103ab Relative to the —+6.5% +7.3% +5.2% +4.2% control FCR   1.598a   1.534b   1.513b   1.521b  1.544b Relative to the — −4.0% −5.3% −4.8% −3.4% control Mortality (%) 11.1a   9.7a   8.3a   6.5a   7.4a ¹Group fed with the basal diet. ²DSM29870/29871/29872 supplemented group fed with the basal diet includingstrain DSM 29870/29871/29872 at 5.10⁸ CFU/kg of feed. ³GalliPro Maxsupplemented group fed with the basal diet including GalliPro Max at8.10⁸ CFU/kg of feed. ⁴Means from 12 replicates of 18 birds per pen foreach group. Raw data (n = 36) were analyzed using variance analysis withblock (n = 12) and treatments (n = 3) as fixed effect. a,b,cValues withdifferent superscripts are significantly (P < 0.05) different from eachother.

In this experiment, the administration of the strains DSM 29870, DSM29871, DSM 29872 (as GalliPro Max) had not significant effect on thefeed intake, but led to the significant improvement of the body weightgain by 6.5%, 7.3% and 5.2%, respectively. GalliPro Max improved alsothe body weight gain (+4.2%), but not significantly.

All NZB strains improved significantly the feed conversion ratio by4.0%, 5.3% and 4.8% (DSM 29870, DSM 29871 and DSM 29872, respectively)as GalliPro Max which reached 3.4% of improvement.

Regarding the mortality level, there were no significant differencesbetween the treatments.

Example 11: Synthesis and Meta-Analysis

The results of the three performance experiments described above aresummarized in Table 11.1.

TABLE 11.1 Results of the individual studies Relative Relative RelativeFI to the BWG to the to the Trial Treatments (g/bird) control (g/bird)control FCR control 1 Control 3245a —  1987ab — 1.633a — DSM 29870 3256a+0.3%  2026ab +2.0% 1.607b −1.6% DSM 29871 3221a −0.7%  2002ab +0.8%1.609b −1.5% DSM 29872 3263a +0.6% 2038a +2.6% 1.602b −2.0% GalliPro Max3203a −1.3% 1973b −0.7%  1.624ab −0.6% 2 Control 3573a — 2361a — 1.513a— DSM 29870 3542a −0.9% 2416b +2.3% 1.467b −3.6% DSM 29871 3606a +0.9% 2401ab +1.7%  1.501bc −0.8% DSM 29872 3515a −1.0% 2424b +2.6% 1.451a−4.2% GalliPro Max 3541a −0.9%  2398ab +1.5%  1.477ab −2.4% 3 Control3217a — 2019b — 1.598a — DSM 29870 3295a +2.4% 2149a +6.5% 1.534b −4.0%DSM 29871 3281a +2.0% 2167a +7.3% 1.513b −5.3% DSM 29872 3230a +0.4%2124a +5.2% 1.521b −4.8% GalliPro Max 3246a +0.9%  2103ab +4.2% 1.544b−3.4% a,b,cValues with different superscripts are significantly (P <0.05) different from each other.

These trials having a similar experimental design, all the data obtainedwere pooled and combined in a meta-analysis (after having been testedfor homogeneity). This meta-analysis involved 3 980 broilers in 160replicates (32 replicates and 796 animals for each experimental group).The results are shown in Table 11.

Data (n=160) were subjected to a mixed model ANOVA, using the GLIMMIXprocedure of SAS (SAS Institute, 2002-2012) to establish differencesbetween diets. Pen was considered as the experimental unit. The modelincluded diets (n=5) as fixed effect and experiment (n=3) as randomeffect. Results are reported as least square means. LS means wereassumed to be different at P≤0.05.

TABLE 11.2 meta-analysis of three experiments showing the effect ofdietary supplementation of the strain DSM 29870, DSM 29871 and DSM 29872on broiler performance GalliPro DSM 29870² DSM 29871² DSM 29872² Max³supplemented supplemented supplemented supplemented Parameters⁴ Control¹group group group group FI (g/bird) 3343a 3360a 3361a 3356a 3347aRelative to the — +0.5% +0.5% +0.4% +0.1% control BWG (g/bird) 2145b2208a 2202ab 2211a 2171ab Relative to the — +2.9% +2.6% +3.0% +1.2%control FCR   1.560a   1.530b   1.53bc   1.52c   1.550ab Relative to the— −2.4% −2.2% −2.7% −1.1% control ¹Group fed with the basal diet ²DSM29870/29871/29872 supplemented group fed with the basal diet includingstrain DSM 29870/29871/29872 at 5.10⁸ CFU/kg of feed. ³GalliPro Maxsupplemented group fed with the basal diet including GalliPro Max at8.10⁸ CFU/kg of feed. ⁴Means from 32 observations for each experimentalgroup (796 animals/group). a,b,cValues with different superscripts aresignificantly (P < 0.05) different from each other.

This meta-analysis showed that the use of the strain DSM 29870, DSM29871 or DSM 29872 in a corn/soybean meal-based diet significantlyimproved in average the body weight gain (+2.9%, +2.6% and +3.0%,respectively) and the feed conversion ratio (−2.4%, −2.2% and −2.7%,respectively) with no effect on the feed intake.

CONCLUSION

In the working examples above, positive effects of strain DSM 29870 havebeen demonstrated on broiler performances (i.e., body weight gain andfeed conversion ratio) fed a corn/soybean meal-based diet.

All the data obtained from the experiments described in the aboveexamples also showed that the effect of strain DSM 29870 on the feedconversion ratio is due to its effect on the body weight. These effectsmight be associated with either health effect or metabolism improvement.

Strain DSM 29870 showed positive and significant effects on body weightgain and feed conversion ratio in 66% and 100% of the working examplesabove, respectively.

GalliPro Max showed no significant effect on the body weight gain in anyof the working examples above and showed significant and positive effecton feed conversion ratio in one third of the experiments.

In the experiments described in the above examples, the better effect ofstrain DSM 29870 compared to GalliPro Max on broilers performance hasbeen demonstrated. The effect observed using DSM 29870 were in averagetwice those observed for GalliPro Max.

Example 12: Determination of Monensin Compatibility

Monensin compatibility of Bacillus strains DSM 29869, DSM 29870, DSM29871, and DSM 29872 was determined using a modified broth microdilution similar to the method described in the Example 5. Briefly, asingle colony of Bacillus spp. (from overnight tryptic soy agar plates)was inoculated into Mueller Hinton Broth (MHB) and cultured overnight.Sterile media was the inoculated with the overnight culture and allowedto grow for 4 hours to test bacteria in log growth phase. Cultures werethen diluted once more 1:200 into fresh MHB and 90 μL of this inoculatedbroth was added to the diluted monensin at the indicated concentrations.Prior art strains were also tested for comparison: NN019785, NN062266(NRRL B-50013), NN062267 (NRRL B-50104), NN062278 (PTA-6507), NN062319(FERM BP-1096), NN062440, NN062441 (DSM 17236), NN062439.

Strains:

Bacillus amyloliquefaciens DSM 29869

Bacillus subtilis DSM 29870

Bacillus subtilis DSM 29871

Bacillus amyloliquefaciens DSM 29872

Bacillus licheniformis NN019785

Bacillus amyloliquefaciens NN062266 (NRRL B-50013)

Bacillus subtilis NN062267 (NRRL B-50104)

Bacillus subtilis NN062278 (PTA-6507)

Bacillus amyloliquefaciens NN062319 (FERM BP-1096)

Bacillus subtilis NN062440

Bacillus licheniformis NN062441 (DSM 17236)

Bacillus amyloliquefaciens NN062439

Materials:

Monensin sodium salt (Sigma, CAS no. 22373-78-0, solubilized in 96%ethanol)

Mueller Hinton Broth (Becton, Dickinson and Company, 275730)

Tryptic soy agar (Becton, Dickinson and Company, 236920)

Micro titer plates: Costar plate, polypropylene, flat bottom, Corning,3628

Borosilicate glass tubes: Kimbale, 16×125 mm, 73500-16125

Adhesive gas permeable seals: Thermo Scientific, AB-0718

Preparation of Bacteria:

Bacillus spp. were grown overnight on tryptic soy agar plates (40 g/L)at 37° C. Mueller Hinton broth (21 g/L) was dissolved in water andautoclaved in glass tubes containing 5 mL of broth each. A single colonyof Bacillus spp. (from overnight plates) was inoculated into MuellerHinton Broth (MHB) and incubated overnight at 37° C. shaking at 200 rpm.A 5 mL glass tube of fresh, sterile media was then inoculated with 25 mLof overnight culture and allowed to grow for 4 hours at 37° C. Cultureswere then diluted once more 1:200 into fresh MHB. 90 μL of thisinoculated broth was then added to the diluted antibiotic at theindicated concentrations.

Preparation of Assay Plates:

Monensin was diluted into 96% ethanol to a concentration of 800 μg/mL.This solution was then diluted 10-fold into sterile phosphate buffer toa concentration of 80 μg/mL. A two fold dilution series was prepared inMHB down to the concentration 2.5 μg/mL. 10 μl of each dilution and ofeach antibiotic was pipetted into a micro titer plate. Later, when theantibiotics were mixed with the suspension of bacteria, the samples werediluted 10× (10 μL sample in a total volume of 100 μl). This resulted inthe final test range of 0.25-8 μg/ml.

90 μl of the bacterial suspensions were added to the assay plates. Theassay plates were then covered with an adhesive glass permeable seal andincubated overnight at 37° C. shaking at 200 rpm. The maximum compatibleconcentration was determined similar to a MIC analysis as theconcentration above that which inhibited 80% of bacteria as detected bythe unaided eye.

Results:

A potential challenge of delivering Bacillus spp. in feed is the commonuse of antibiotics as growth promoters in feed. Therefore it isnecessary to determine the compatibility of strains with commonly-usedfeed antibiotics in order to identify any potential conflicts with useas a direct fed microbial. Therefore, the monensin compatibility ofBacillus strains DSM 29869, DSM 29870, DSM 29871, and DSM 29872 weredetermined along with prior art strains. Bacillus strains DSM 29869, DSM29870, DSM 29871, and DSM 29872 indicated a higher level ofcompatibility with monensin than most of the prior art strains includedherein: NN019785, NN062266 (NRRL B-50013), NN062267 (NRRL B-50104),NN062278 (PTA-6507), NN062319 (FERM BP-1096), NN062440, NN062441 (DSM17236), NN062439.

TABLE 12.1 Monensin compatibility results Monensin Species Product Name(μg/mL) NN062677 DSM29870 Bacillus subtilis 2.7 NN062673 DSM29871Bacillus subtilis 2.0 NN062683 DSM29872 Bacillus amyloliquefaciens 4.0NN062676 DSM29869 Bacillus amyloliquefaciens 2.0 NN019785 Bacilluslicheniformis BioPlus 2B (Chr. Hansen) 0.8 NN062266 NRRL B- Bacillusamyloliquefaciens Eviva Pro (Dupont) 1.1 50013 NN062267 NRRL B- Bacillussubtilis Eviva Pro (Dupont) 1.1 50104 NN062278 PTA-6507 Bacillussubtilis Eviva Pro (Dupont) 1.4 NN062319 FERM BP- Bacillusamyloliquefaciens Calsporin (Calpis) 2.2 1096 NN062440 Bacillus subtilisGalliPro Max (Chr. Hansen) 0.4 NN062441 DSM Bacillus licheniformisGalliPro Tect (Chr. Hansen) 0.9 17236 NN062439 Bacillusamyloliquefaciens Clostat (Kemin) 2.1

What is claimed is:
 1. A composition comprising a carrier, a flowabilityagent comprising sodium aluminium silicate and/or colloidal amorphoussilica, and spores of a Bacillus strain characterized in having depositaccession number DSM 29870 or a mutant strain thereof having all of theidentifying characteristics of Bacillus DSM 29870; wherein the Bacillusstrain is sensitive to at least seven of the antibiotics selected fromthe group consisting of Vancomycin, Clindamycin, Chloramphenicol,Gentamicin, Kanamycin, Streptomycin, Erythromycin and Tetracycline; andwherein the Bacillus strain is non-hemolytic.
 2. The compositionaccording to claim 1, wherein the Bacillus spores of the composition arepresent as dried spores.
 3. The composition according to claim 2,wherein the composition has a bacterial count of each Bacillus sporebetween 1×10⁴ and 1×10¹⁸ CFU/kg of composition.
 4. The compositionaccording to claim 1, wherein the carrier comprises one or more of thefollowing compounds: water, glycerol, ethylene glycol, 1, 2-propyleneglycol or 1, 3-propylene glycol, sodium chloride, sodium benzoate,potassium sorbate, sodium sulfate, potassium sulfate, magnesium sulfate,sodium thiosulfate, calcium carbonate, sodium citrate, dextrin,maltodextrin, glucose, sucrose, sorbitol, lactose, wheat flour, wheatbran, corn gluten meal, starch, farigel, cassava cores, Sipernat 50S,polyethylene glycol 200, polyethylene glycol 400, polyethylene glycol600, polyethylene glycol 1000, polyethylene glycol 1500, polyethyleneglycol 4000, carbopol, and cellulose.
 5. The composition according toclaim 1, wherein the carrier comprises calcium carbonate.
 6. Thecomposition according to claim 1, further comprising one or morecomponents selected from the list consisting of: one or more enzymes,one or more additional microbes, one or more vitamins, one or moreminerals, one or more amino acids, and one or more other feedingredients.
 7. A composition comprising a carrier, a flowability agentcomprising sodium aluminium silicate and/or colloidal amorphous silica,and spores of a Bacillus strain characterized in having depositaccession number DSM 29869 or a mutant strain thereof having all of theidentifying characteristics of Bacillus DSM 29869, wherein the Bacillusstrain is sensitive to at least seven of the antibiotics selected fromthe group consisting of Vancomycin, Clindamycin, Chloramphenicol,Gentamicin, Kanamycin, Streptomycin, Erythromycin and Tetracycline; andwherein the Bacillus strain is non-hemolytic.
 8. The compositionaccording to claim 7, wherein the Bacillus spores of the composition arepresent as dried spores.
 9. The composition according to claim 8,wherein the composition has a bacterial count of each Bacillus sporebetween 1×10⁴ and 1×10¹⁸ CFU/kg of composition.
 10. The compositionaccording to claim 7, wherein the carrier comprises one or more of thefollowing compounds: water, glycerol, ethylene glycol, 1, 2-propyleneglycol or 1, 3-propylene glycol, sodium chloride, sodium benzoate,potassium sorbate, sodium sulfate, potassium sulfate, magnesium sulfate,sodium thiosulfate, calcium carbonate, sodium citrate, dextrin,maltodextrin, glucose, sucrose, sorbitol, lactose, wheat flour, wheatbran, corn gluten meal, starch, farigel, cassava cores, Sipernat 50S,polyethylene glycol 200, polyethylene glycol 400, polyethylene glycol600, polyethylene glycol 1000, polyethylene glycol 1500, polyethyleneglycol 4000, carbopol, and cellulose.
 11. The composition according toclaim 7, further comprising one or more components selected from thelist consisting of: one or more enzymes, one or more additionalmicrobes, one or more vitamins, one or more minerals, one or more aminoacids, and one or more other feed ingredients.
 12. A compositioncomprising a carrier, a flowability agent comprising sodium aluminiumsilicate and/or colloidal amorphous silica, and spores of a Bacillusstrain characterized in having deposit accession number DSM 29871 or amutant strain thereof having all of the identifying characteristics ofBacillus DSM 29871, wherein the Bacillus strain is sensitive to at leastseven of the antibiotics selected from the group consisting ofVancomycin, Clindamycin, Chloramphenicol, Gentamicin, Kanamycin,Streptomycin, Erythromycin and Tetracycline; and wherein the Bacillusstrain is non-hemolytic.
 13. The composition according to claim 12,wherein the Bacillus spores of the composition are present as driedspores.
 14. The composition according to claim 13, wherein thecomposition has a bacterial count of each Bacillus spore between 1×10⁴and 1×10¹⁸ CFU/kg of composition.
 15. The composition according to claim12, wherein the carrier comprises one or more of the followingcompounds: water, glycerol, ethylene glycol, 1, 2-propylene glycol or 1,3-propylene glycol, sodium chloride, sodium benzoate, potassium sorbate,sodium sulfate, potassium sulfate, magnesium sulfate, sodiumthiosulfate, calcium carbonate, sodium citrate, dextrin, maltodextrin,glucose, sucrose, sorbitol, lactose, wheat flour, wheat bran, corngluten meal, starch, farigel, cassava cores, Sipernat 50S, polyethyleneglycol 200, polyethylene glycol 400, polyethylene glycol 600,polyethylene glycol 1000, polyethylene glycol 1500, polyethylene glycol4000, carbopol, and cellulose.
 16. The composition according to claim12, further comprising one or more components selected from the listconsisting of: one or more enzymes, one or more additional microbes, oneor more vitamins, one or more minerals, one or more amino acids, and oneor more other feed ingredients.
 17. A composition comprising a carrier,a flowability agent comprising sodium aluminium silicate and/orcolloidal amorphous silica, and spores of a Bacillus straincharacterized in having deposit accession number DSM 29872 or a mutantstrain thereof having all of the identifying characteristics of BacillusDSM 29872, wherein the Bacillus strain is sensitive to at least seven ofthe antibiotics selected from the group consisting of Vancomycin,Clindamycin, Chloramphenicol, Gentamicin, Kanamycin, Streptomycin,Erythromycin and Tetracycline; and wherein the Bacillus strain isnon-hemolytic.
 18. The composition according to claim 17, wherein theBacillus spores of the composition are present as dried spores.
 19. Thecomposition according to claim 17, wherein the composition has abacterial count of each Bacillus spore between 1×10⁴ and 1×10¹⁸ CFU/kgof composition.
 20. The composition according to claim 17, wherein thecarrier comprises one or more of the following compounds: water,glycerol, ethylene glycol, 1, 2-propylene glycol or 1, 3-propyleneglycol, sodium chloride, sodium benzoate, potassium sorbate, sodiumsulfate, potassium sulfate, magnesium sulfate, sodium thiosulfate,calcium carbonate, sodium citrate, dextrin, maltodextrin, glucose,sucrose, sorbitol, lactose, wheat flour, wheat bran, corn gluten meal,starch, farigel, cassava cores, Sipernat 50S, polyethylene glycol 200,polyethylene glycol 400, polyethylene glycol 600, polyethylene glycol1000, polyethylene glycol 1500, polyethylene glycol 4000, carbopol, andcellulose.
 21. The composition according to claim 17, further comprisingone or more components selected from the list consisting of: one or moreenzymes, one or more additional microbes, one or more vitamins, one ormore minerals, one or more amino acids, and one or more other feedingredients.